Therapeutic combinations of an irreversible BTK inhibitor and a macrocyclic reversible BTK inhibitor

Abstract

The present invention relates to therapeutic combinations of an irreversible BTK inhibitor and a macrocyclic reversible BTK inhibitor and a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject receives or has received a Bruton's tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, using said therapeutic combinations. The present invention further relates to method treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject has previously been treated with an irreversible BTK inhibitor, using said therapeutic combinations. In particular embodiments the method comprises: a) monitoring the patient over the course of therapy to determine whether the subject has a C481 mutation in BTK and b) co-administering to the patient an irreversible and a macrocyclic reversible BTK inhibitor if the patient has a C481 mutation in BTK. The invention further relates to a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, wherein the treatment comprises administering said therapeutic combinations.

Description

[0001] Therapeutic combinations of an irreversible BTK inhibitor and a macrocyclic reversible BTK inhibitor

[0002] Field of the invention

[0003] The present invention relates to therapeutic combinations of an irreversible BTK inhibitor and a macrocyclic reversible BTK inhibitor and relates to a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, using said therapeutic combinations. The present invention further relates to a method of treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject has previously been treated with an irreversible BTK inhibitor, using said therapeutic combinations. In particular embodiments the method comprises: a) monitoring the patient over the course of therapy to determine whether the subject has a C481 mutation in BTK and b) co- administering to the patient an irreversible and a macrocyclic reversible BTK inhibitor if the patient has a C481 mutation in BTK. The invention further relates to a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, wherein the treatment comprises administering said therapeutic combinations.

[0004] Background of the invention

[0005] Kinases are enzymes that transfer a phosphate group from ATP to a protein. Kinases play an important role in regulating cellular functions such as cell proliferation, subcellular translocation, apoptosis, inflammation and metabolism (Attwood M.M. et al (2021 ) Nat Rev Drug Discov). The human kinome is composed of over 500 kinases. The development of small- molecule kinase inhibitors for the treatment of diverse types of cancer has proven successful in clinical therapy.

[0006] Bruton’s tyrosine kinase (BTK) is a member of the Src-related Tec family of protein kinases constituting a large subset of kinases, which play a central role in the regulation of a wide variety of cellular signaling processes. BTK plays a key role in B-cell receptor (BCR) signaling and a critical role in the regulation of survival, proliferation, activation and differentiation of B-lineage cells. Targeting of BTK with small molecule inhibitors such as the FDA approved covalent, irreversible, BTK inhibitors ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib has proven to be efficacious in several B-cell malignancies including Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), Waldenstrom’s Macroglobulinemia (WM) and Small Lymphocytic Lymphoma SLL.

[0007] Where tumors have a strong oncogenic drive from one particular kinase, specific amino acid mutations have been observed as acquired resistance mechanisms of targeted drug molecules. These mutations may naturally occur in very low levels but become more prevalent upon drug treatment (pre-existing mutations), or these can also be produced by random mutation within tumors (acquired mutations) (Barouch-Bentov et al. 2011). Where such mutations have a clear advantage upon drug treatment ( / .e. a mutation where the drug is no longer able to bind to the target) then resistance may rapidly develop and patients can relapse quickly. In these cases, the understanding of the impact and mechanism of these mutations has enabled additional drug discovery efforts to develop subsequent “generations” of drug molecules to target such resistance forms.

[0008] A drawback of the first generation BTK inhibitor, ibrutinib, is that drug resistance in 13- cell malignancies can develop when BTK acquires mutations at the cysteine at position C481 of the kinase domain. This mutation abrogates the covalent binding of ibrutinib hampering its efficacy. Quinquenel et al. performed a ‘snapshot’ screening to determine the prevalence of resistance mutations and found that the presence of the BTK mutation was significantly associated with subsequent CLL progression. The correlation between disease progression, and the emergence and temporal dynamics of the most common resistance inducing C481S BTK mutation have been determined for CLL patients receiving single-agent ibrutinib treatment (Bodor et al. British Journal of Haematology 2021). Using digital droplet PCR the authors have been able to show that in 72.7% of the patients emergence of the BTK C481S mutation preceded the symptoms of clinical relapse with a median of nine months. Woyach et al. showed that that detection of the C481 mutation in patients being treated with covalent, irreversible, BTK inhibitors is an indication for imminent relapse, usually within 12-18 months after detection (Woyach et al. (2017) J. Clin. Oncol. 35, 1437-1443). Since ibrutinib resistance confers poor survival, early detection of resistance provides clinically relevant information for the transition of affected patients to alternative treatment strategies.

[0009] Second-generation covalent, irreversible, BTK inhibitors, which include acalabrutinib, zanubrutinib, and tirabrutinib, offer greater BTK selectivity and therefore limited off-target toxicity. These inhibitors, however, do not overcome resistance by C481 mutation. To treat patients with relapsed CLL having C481 BTK mutations non-covalent, reversible, BTK inhibitors have been developed including LOXO-305 (pirtobrutinib) and ARQ-531 (nemtabrutinib) and vecabrutinib. These agents do not require binding to the BTK C481 residue and effectively inhibit both wild type and mutant BTK with C481 substitutions. In preclinical studies, non-covalent BTK inhibitors including pirtobrutinib (LOXO-305), ARQ-351 and vecabrutinib, inhibited B-cell-receptor signaling in BTK C481 -mutant cell and animal models. Moreover, the phase 1 -2 clinical trial of pirtobrutinib showed promising efficacy for patients with B-cell cancer who had previously been treated with covalent BTK inhibitors (with 62% of patients with CLL having a response), including patients with or without BTK C481 mutations (with a response occurring in 71 % and 66% of the patients, respectively) (Wang et al. (2022) N. Engl. J. Med. 386, 735-43).

[0010] Pirtobrutinib was first disclosed in WO2017 / 10361 1 , ARQ-531 was disclosed in WO2017 / 111787 and vecabrutinib is disclosed in WO2013 / 185084. Further reversible BTK inhibitors are disclosed in WO2017 / 046604, , W02020 / 015735, WO2020 / 239124, WO2021 / 093839, W02020 / 043638, WO2013 / 067274, WO2018097234, WO2013 / 010380, W02016 / 161570, W02016 / 161571 , W02016 / 106624, W02016 / 106625, W02016 / 106626, W02016106623, W02016 / 106628 and W02016 / 109222.

[0011] Furthermore other reversible BTK inhibitors in development are fenebrutinib (Crawford et al. 2018 and Reiff et al. 2018), BMS-986142 (Watterson et al. 2016), luxeptinib (Thieme et al. 2022 and WO2014 / 104757, AS-0871 (Kawahata et al. 2018 and W02015 / 012149), AS-1763 (Kawahata et al. 2021 and WO2018 / 097234) and BIIB091 (Hopkins et al. 2021 and WO2018 / 191577). These reversible BTK inhibitors are being developed not only for B-cell hematological malignancies, but also for immunological and inflammatory diseases.

[0012] Despite the successful treatment of patients with a C481 mutation with pirtobrutinib new acquired mutations have emerged after treatment with this non-covalent inhibitor. Mutations were identified in these patients that were clustered in the kinase domain of BTK (V416L, A428D, M437R, T474I, and L528W) and that conferred resistance to pirtobrutinib and other non- covalent, reversible, inhibitors.

[0013] The most well-known clinical documented BTK mutations for ibrutinib, acalabrutinib, zanubrutinib and pirtobrutinib are given in Figure 1 mutants (Nakoda et al. 2023).

[0014] A general desire of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance (Al-Lazikani et al. 2012; Yap et al. 2013 and Li et al. 2014) Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. Cancer cell lines are an attractive model to investigate new drug combinations because they can be used to determine whether new combinations are truly synergistic, as opposed to additive (Zhao et al. 2004 and Chou 2010). Moreover, cancer cell lines provide a good representation of the diversity of genetic changes that drive human cancers (Garnett et al. 2012 and Barretina et al. 2013).

[0015] There are several ways to determine synergy between two compounds in an in vitro assay. From this, two basic methods have evolved, the curve shift analysis (Chou 2010 and Straetemans et al. 2005) and the combination matrix experiment with Bliss-scoring (Borisy et al. 2003; Cokol et al. 2011 and Mathews et al. 2014).

[0016] Summary of the invention

[0017] In a first aspect of the invention, there is provided a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, the method comprising the steps: a) monitoring the subject, preferably at predetermined intervals of time, over the course of the therapy for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor of the (mono) therapy, preferably the subject has a mutation in an endogenous gene encoding BTK that results in expression of a BTK protein with a modification at an amino acid position corresponding to amino acid position 481 of the amino acid sequence according to SEQ ID NO: I; and

[0018] (b) if the subject has shown said mutation, administering to the subject a therapeutically combinatory amount of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular if the subject has the BTK modification corresponding to amino acid position 481 identified in step a), wherein the reversible BTK inhibitor is a compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt and / or solvate thereof.

[0019] In another aspect of the invention, there is provided a method for treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has previously been treated with an irreversible BTK inhibitor, and wherein the method comprises administering to said subject a therapeutic combinatory amount of an irreversible BTK inhibitor and a reversible BTK inhibitor, wherein the reversible BTK inhibitor is a compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt and / or solvate thereof.

[0020] In another aspect of the invention, there is provided a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, the method comprising: administering to the subject a therapeutic combinatory amount of an irreversible BTK inhibitor and a reversible BTK inhibitor, wherein the reversible BTK inhibitor is a compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt and / or solvate thereof.

[0021] In other aspects of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method according to the invention.

[0022] In a particular aspect of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, the method comprising the steps: a) monitoring the subject, preferably at predetermined intervals of time, over the course of the therapy for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor of the (mono) therapy, preferably the subject has a mutation in an endogenous gene encoding BTK that results in expression of a BTK protein with a modification at an amino acid position corresponding to amino acid position 481 of the amino acid sequence according to SEQ ID NO: I; and

[0023] (b) if the subject has shown said mutation, administering to the subject a therapeutically combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor, in particular if the subject has the BTK modification corresponding to amino acid position 481 identified in step a) wherein the reversible BTK inhibitor is a compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt and / or solvate thereof.

[0024] In a particular aspect of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has previously been treated with an irreversible BTK inhibitor, and wherein the method comprises administering to said subject a therapeutic combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor wherein the reversible BTK inhibitor is a compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt and / or solvate thereof.

[0025] In a particular aspect of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, the method comprising: administering to the subject a therapeutic combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor, wherein the reversible BTK inhibitor is a compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt and / or solvate thereof.

[0026] In other aspects of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor according to the invention.

[0027] In other aspects of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor according to the invention for use as a medicament.

[0028] In other aspects of the invention, there is provided use of a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor according to the invention in the manufacture of a medicament for treating a subject having a hyperproliferative disease, preferably treating a subject having a B-cell hematological malignancy

[0029] The present inventors have surprisingly found that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular a macrocyclic BTK inhibitor according to the invention, is more effective than an irreversible or reversible BTK inhibitor alone.

[0030] In particular, it has been shown that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular the macrocyclic BTK inhibitor according to the invention, is more effective than an irreversible or reversible BTK inhibitor alone in inhibiting B-cell lymphoma cells comprising two separate cell pools each containing a different mutation in BTK.

[0031] Moreover, combinations of a macrocyclic reversible BTK inhibitor compound according to the invention and an irreversible, covalent, inhibitor is synergistically effective in inhibiting B-cell lymphoma cells. In specific examples, a synergistic effect of inhibition has been shown in a B- cell lymphoma cell line in which BTK has at least a C481S modification.

[0032] In another aspect, there is provided a pharmaceutical composition comprising a combination of an irreversible BTK inhibitor and a macrocyclic reversible BTK inhibitor, wherein the macrocyclic reversible BTK inhibitor is a compound according to any one of Formula (l-a) to (l-h), in a particular example according to a compound of Formula (A).

[0033] The present invention will be illustrated further by means of the following non-limiting examples.

[0034] Definitions

[0035] The term "pharmaceutical composition” as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.

[0036] The term "pharmaceutically acceptable” as used herein has its conventional meaning and refers to compounds, material, compositions and / or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit / risk ratio.

[0037] The term "effective amount' as used herein, refers to an amount of the compound of the invention, and / or an additional therapeutic agent, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventive effect when administered to a subject suffering from a BTK-mediated disease or disorder. In the combination therapies of the present invention, as effective amount can refer to each individual agent or to the combination as a whole, wherein the amounts of all agents administered are together effective, but wherein the component agent of the combination may not be present individually in an effective amount. The term "therapeutically combinatory amount” as used herein has its meaning in the context of combination therapy of the compounds according to the invention, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventive effect when administered to a subject suffering from a hyperproliferative disease, in particular a B-cell hematological malignancy . In particular embodiments, the therapeutically combinatory amount is the total amount of the two (or more) compounds selected of an irreversible BTK inhibitor and a reversible BTK inhibitor used for treating the hyperproliferative disease, in particular a B-cell hematological malignancy.

[0038] The term “combination” as used herein, means a product that results from the mixing or combining of an irreversible BTK inhibitor and a reversible BTK inhibitor (and any additional therapeutic agents) and includes both fixed and non-fixed combinations. The term “fixed combination” means that the irreversible BTK inhibitor and the reversible BTK inhibitor are both administered in a single entity or dosage form. The term “non-fixed combination” means that the irreversible BTK inhibitor and the reversible BTK inhibitor are administered as separate entities or dosage forms either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients.

[0039] A "subject' is a human or non-human mammal. In one embodiment, a subject is a human.

[0040] The term "controlling” is intended to refer to all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of the diseases and conditions affecting the mammal. However, "controlling” does not necessarily indicate a total elimination of all disease and condition symptoms, and is intended to include prophylactic treatment.

[0041] The term "excipient” as used herein has its conventional meaning and refers to a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing a granulate, solid or liquid oral dosage formulation.

[0042] The term "salt” as used herein has its conventional meaning and includes the acid addition and base salts of the compound of the invention.

[0043] The term "solvate” as used herein has its conventional meaning. One or more compounds of the invention or the pharmaceutically acceptable salts thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. "Solvate" means a physical association of a compound of this invention with one or more solvent molecules. This physical association Involves varying degrees of ionic and covalent bonding. Including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Examples of suitable solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate wherein the solvent molecule is H2O and includes any hydrate of the compound or the salt of said compound. The term "treatment” as used herein has its conventional meaning and refers to curative, disease controlling, palliative and prophylactic treatment.

[0044] The term "unit dosage form” has its conventional meaning and refers to a dosage form which has the capacity of being administered to a subject, preferably a human, to be effective, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising the therapeutic agent, i.e. the compound of the invention.

[0045] The term "BTK” as used herein has its conventional meaning and refers to Bruton's Tyrosine Kinase. Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein kinases which are a large subset of kinases which play a central role in the regulation of a wide variety of cellular signaling processes. BTK plays a key role in the B-cell receptor signaling and a critical role in the regulation of survival, proliferation, activation and differentiation of B- lineage cells. Targeting of BTK with small molecule inhibitors such as the FDA approved irreversible BTK inhibitors ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib has proven to be efficacious in several B-cell malignancies including Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), Waldenstrom’s Macroglobulinemia (WM) and Small Lymphocytic Lymphoma SLL. Combinations of BTK inhibitors with other novel drugs or regimens results in more profound responses and much higher rates of minimal residual disease negativity.

[0046] The term "BTK inhibitor” as used herein has its conventional meaning and refers to an inhibitor for BTK. A BTK inhibitor may be a small molecule inhibitor. Inhibitors may be irreversible inhibitors, such as by forming a covalent bond, and may be reversible inhibitors, which may form a reversible interaction with BTK.

[0047] The term “covalent BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor that reacts with its target protein (BTK) to form a covalent complex in which the protein has lost its function. Covalent inhibitors can be reversible or irreversible, depending on the rate of the reverse reaction. The terms 'covalent inhibitor' and 'irreversible inhibitor' are often used and are considered the same for the purpose of this patent application .

[0048] The term "irreversible BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor that, after formation of a covalent complex with the protein, possesses an off-rate that is slow relative to the rate of re-synthesis of the target protein (BTK) in vivo, so that once the target protein is inhibited, it does not regain activity.

[0049] The term “rate of re-synthesis” as used herein has its conventional meaning and refers to the rate at which a cell and / or organism replaces a protein target with freshly synthesized functional protein. The re-synthesis rate defines the rate at which an irreversibly inhibited protein target will recover activity in vivo, once the inhibitor is no longer present.

[0050] The term "reversible BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor that inactivates the BTK enzyme through non-covalent, reversed, interactions. Unlike an irreversible inhibitor, a reversible inhibitor can dissociate from the enzyme.

[0051] The term "mutant-BTK” as used herein has its conventional meaning and refers to mutations of BTK. Mutations of BTK may be referred to by an altered or modified amino acid target (such as C as single-letter amino acid code for cysteine) at a certain position of the BTK structure (such as 481). Additionally, the amino acid substitution at the modification position may be referred to by an additional amino acid single-letter amino acid code, such as C481S for serine substitution and C481 T for threonine substitution of cysteine at the 481 position.

[0052] The term “ modification" as used herein has its conventional meaning and refers to modification of a sequence of amino acids of a polypeptide, protein or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively.

[0053] The term "resistance to BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor, which shows a reduction in effectiveness, after being effective initially in treating a hyperproliferative disease, in particular a B-cell hematological malignancy.

[0054] A drawback of the currently approved irreversible inhibitors is that patients treated with these inhibitors can develop drug resistance when proteins with variations at the catalytic site are not able to bind efficiently to irreversible inhibitors in patients. This is a rather common event in patients treated with irreversible inhibitors and who experience relapse. A major mechanism for the acquired resistance is the emergence of BTK cysteine 481 (C 481) mutations. These mutations hamper binding of irreversible inhibitors such as ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib which form a covalent bond with this amino acid. Other mutations / modifications that can result in acquired resistance of irreversible covalent and reversible non-covalent BTK inhibitors are valine 416 (V416), alanine 428 (A428), methionine 437 (M437), threonine 474 (T474) and leucine 528 (L528) modifications, which can reduce BTK inhibitor binding to BTK.

[0055] By the term “drug resistance" as used herein is meant the major cause of cancer treatment failure. While a treatment may be effective initially, the heterogeneity of cancer and its ability to adapt can allow the cancer to become resistant to the treatment and regrow.

[0056] The term "relapse” as used herein has the conventional meaning and refers to evidence of disease progression in a patient who has previously achieved criteria of a complete response or partial remission.

[0057] The term "disease progression” as used herein has the conventional meaning and refers to a measured increase in tumor size or tumor burden.

[0058] The term “ recurrent or refractory form of a hyperproliferative disease” as used herein has the conventional meaning and refers to a particular cancer that is resistant, or non-responsive to therapy with a particular therapeutic agent. A cancer can be refractory to therapy with a particular therapeutic agent either from the onset of treatment (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.

[0059] The term "acquired drug resistance” as used herein has the conventional meaning and refers to resistance of a drug caused by mutations in the target protein following treatment. The mutations in the target protein hamper the binding of drug resulting in a regrowth of the tumor The term "clinical drug resistance” as used herein has the conventional meaning and refers to growth of a tumour while the patient is on treatment, that develops following after an initial clinical benefit (a clinical response or prolonged stable disease).

[0060] The term "wt-BTK” or “WT-BTK” or “BTKWT" as used herein has its conventional meaning and refers to wild-type Bruton’s Tyrosine Kinase. A wild-type BTK has the regular meaning of a phenotype of the typical form of BTK as it occurs in nature. Originally, the wild-type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele.

[0061] The term "macrocycle” as used herein has its conventional meaning and refers to a part of a molecule containing a ring consisting of 12 or more ring atoms forming said ring. In an example, a twelve membered ring consist of 12 atoms forming said ring.

[0062] The term "binding affinity (KD)” as used herein has its conventional meaning and refers to the equilibrium dissociation constant which is an inverse measure of the affinity of a protein- ligand (small molecule) pair under equilibrium conditions. The value of KDis mathematically equivalent to the ratio koff / kon(or kd / ka) measured using Surface Plasmon Resonance (SPR).

[0063] The term "association rate constant” or “on-rate (konor ka)” as used herein has its conventional meaning and refers to a second-order rate constant that quantifies the rate at which a free ligand and free protein combine (through collisional encounters) to form a binary proteinligand complex.

[0064] The term "dissociation rate constant” or “off-rate ( koffor kd)” as used herein has its conventional meaning and refers to a first-order rate constant that quantifies the rate at which a binary protein-ligand complex dissociates to the free ligand and free protein.

[0065] The term "target residence time tau (τ)” as used herein has its conventional meaning and refers to the time a compound resides on its target. Target residence time (τ) can be determined according to the method as described below in the experimental section.

[0066] The term “IC50” as used herein has its conventional meaning and refers to the concentration of a substance that results in a 50% effect on some measure of biochemical and / or cellular function or substance-target binding interaction.

[0067] The term “pIC50” as used herein has its conventional meaning and refers to the negative logarithm of the IC50in molar concentration.

[0068] The term “GI50” as used herein has its conventional meaning and refers to the concentration of a substance that inhibits cell growth by 50%.

[0069] The term “LD50” as used herein refers to the concentration of a substance that results in 50% cell death.

[0070] The term “efficacy" as used herein has its conventional meaning and refers to the effect that is reached at a particular concentration of a substance.

[0071] A bicyclic ringsystem, as used herein, refers to heterocyclic (heterocyclyl) groups, to cyclic groups having carbon groups only, i.e. without hetero atoms, within the cycle, and to combinations of a heterocyclic (heterocyclyl) group and a cyclic group having carbon groups only, i.e. without hetero atoms, within the cycle.

[0072] A monocylic ringsystem, as used herein, refers both to a heterocyclic (heterocyclyl) group, and to a cyclic group having carbon groups only, i.e. without hetero atoms, within the cycle.

[0073] A heterocyclic (heterocyclyl) group, as used herein, refers to both heteroaryl groups and heterocycloalkyl groups.

[0074] A heterobicyclic group, as used herein, refers to a bicyclic group having one or more heteroatoms, which is saturated, partially unsaturated or unsaturated.

[0075] As used herein, aromatic groups (or aryl groups) include aromatic carbocyclic ring systems (e.g. phenyl) and fused polycyclic aromatic ring systems (e.g. naphthyl and 1 , 2,3,4- tetrahydronaphthyl).

[0076] The term "alkyl," as used herein, refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced with a bond having the specified number of carbon atoms. In different embodiments, an alkyl group contains, for example, from 1 to 6 carbon atoms (1 - 6C) Alkyl or from 1 to 3 carbon atoms (1 -3C)Alkyl. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl and neohexyl. In one embodiment, an alkyl group is linear. In another embodiment, an alkyl group is branched.

[0077] Unless specified otherwise, "alkyl" includes both branched- and straight-chain saturated aliphatic hydrocarbon groups, including all isomers, having the specified number of carbon atoms; for example, "(1 -6C)Alkyl" includes all of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl. "Alkylene" refers to both branched- and straight-chain saturated aliphatic hydrocarbon groups, including all isomers, having the specified number of carbons, and having two terminal end chain attachments; for example, the term "A-C4alkylene-B" represents, for example, A-CH2-CH2-CH2-CH2-B, A-CH2- CH2-CH(CH3)-CH2-B, A-CH2-CH(CH2CH3)-B, A-CH2-C(CH3)(CH3)-B, and the like.

[0078] The term "alkylcarbonyl," as used herein, refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced with a bond attached to a carbonyl group, wherein the aliphatic hydrocarbon group has the specified number of carbon atoms. In different embodiments, an alkyl group or aliphatic hydrocarbon group contains, for example, from 1 to 6 carbon atoms (1 -6C)Alkyl or from 1 to 3 carbon atoms (1 -3C)Alkyl. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl and neohexyl. In one embodiment, an alkyl group is linear. In another embodiment, an alkyl group is branched.

[0079] Cycloalkyl means a cycloalkyl group having the recited number of carbon atoms, with the same meaning as previously defined, such as cyclopropyl, cyclobutyl, or cyclopentyl. "Cycloalkyl" refers to a cycloalkyl-group represented by an indicated number of carbon atoms; for example "(3-6C)cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Heterocycloalkyl means a cycloalkyl group having the recited number of carbon atoms, and 1 -3 heteroatoms selected from N, O and / or S, with the same meaning as previously defined.

[0080] Haloalkyl means a branched or unbranched alkyl group having the recited number of carbon atoms, in which one and up to all hydrogen atoms are replaced by a halogen; halogen is as defined herein. Examples of such branched or straight chained haloalkyl groups useful in the present invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl and n- butyl substituted independently with one or more halogens, e.g., fluoro, chloro, bromo and iodo.

[0081] For example, a halo(1 -3C)alkyl means a branched or unbranched alkyl group having 1 ,2, or 3 carbon atoms, in which at least one hydrogen atom is replaced by a halogen. Examples of "haloalkyl" include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 1 - fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, and perfluoro-n-propyl.

[0082] Alkoxy means an alkoxy group having the recited number of carbon atoms, the alkyl moiety having the same meaning as previously defined, e.g., "Alkoxy" refers to an alkyl-O-group represented by a linear or branched alkyl group of indicated number of carbon atoms attached through an oxygen bridge; for example "(1 -6C)Alkoxy" includes -OCH3, -O-CH2CH3, - OCH(CH3)2, -O(CH2)5CH3, and the like.

[0083] Cycloalkoxy means a cycloalkyl group having the recited number of carbon atoms, with the same meaning as previously defined, attached via a ring carbon atom to an exocyclic oxygen atom, such as cyclopropoxyl, cyclobutoxyl.or cyclopentoxyl. "Cycloalkoxy" refers to a cycloalkyl- O-group represented by a cycloalkyl group of indicated number of carbon atoms attached through an oxygen bridge; for example "(3-6C)cycloalkoxy" includes cyclopropyl-O-, cyclobutyl- O-, cyclopentyl-O-, or cyclohexyl-O-.

[0084] Heterocycloalkoxy means a cycloalkyl group having the recited number of carbon atoms, and 1 -3 heteroatoms selected from N, O and / or S, with the same meaning as previously defined, attached via a ring carbon atom to an exocyclic oxygen atom.

[0085] Unless otherwise specifically noted as only "unsubstituted" or only "substituted", alkyl groups are unsubstituted or substituted with 1 to 3 substituents on each carbon atom.

[0086] It should be noted that any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples and tables herein is assumed to have the sufficient number of hydrogen atom(s) to satisfy the valences.

[0087] The terms first, second, third and the like in the description and in the claims, are used for distinguishing between for example similar elements, compositions, constituents in a composition, or separate method steps, and not necessarily for describing a sequential or chronological order. The terms are interchangeable under appropriate circumstances and the embodiments of the invention can operate in other sequences than described or illustrated herein, unless specified otherwise.

[0088] Furthermore, the various embodiments, although referred to as “preferred” or “e.g.” or “for example” or “in particular” and the like are to be construed as exemplary manners in which the invention may be implemented rather than as limiting the scope of the invention. The term “comprising", used in the claims, should not be interpreted as being restricted to for example the elements or the method steps or the constituents of a compositions listed thereafter; it does not exclude other elements or method steps or constituents in a certain composition. It needs to be interpreted as specifying the presence of the stated features, integers, (method) steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a method comprising steps A and B” should not be limited to a method consisting only of steps A and B, rather with respect to the present invention, the only enumerated steps of the method are A and B, and further the claim should be interpreted as including equivalents of those method steps. Thus, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting only of components A and B, rather with respect to the present invention, the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components.

[0089] In addition, reference to an element or a component by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element or component are present, unless the context clearly requires that there is one and only one of the elements or components. The indefinite article "a" or "an" thus usually means "at least one".

[0090] Legend to the figures

[0091] Figure 1 : Map of clinically documented BTK mutants.

[0092] Figure 2: Proliferation assay of single compounds (monotherapy) in wt-BTK TMD8 cells.

[0093] Figure 3: Proliferation assay of single compounds (monotherapy) in BTK C481S TMD8 cells.

[0094] Figure 4: Proliferation assay of single compounds (monotherapy) in BTK T474I TMD8 cells.

[0095] Figure 5: Proliferation assay of single compounds (monotherapy) in BTK T474I / C481S TMD8 cells.

[0096] Figure 6: Proliferation assay of single compounds (monotherapy) in BTK V416L TMD8 cells.

[0097] Figure 7: Proliferation assay of single compounds (monotherapy) in BTK L528W TMD8 cells.

[0098] Figure 8: Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells.

[0099] Figure 9: Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells.

[0100] Figure 10: Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells. Figure 1 1 : Proliferation assay of ibrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells.

[0101] Figure 12: Proliferation assay of ibrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells.

[0102] Figure 13: Proliferation assay of ibrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells.

[0103] Figure 14: Proliferation assay of acalabrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells.

[0104] Figure 15: Proliferation assay of acalabrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells.

[0105] Figure 16: Proliferation assay of acalabrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells.

[0106] Figure 17A - 17G : Proliferation assay of ibrutinib and Compound A (combination therapy) in BTK C481S TMD8 cells (curve shift analysis).

[0107] Figure 18: Excess over Bliss score matrix of Compound A and ibrutinib in BTK C481S TMD8 cells.

[0108] Figure 19A - 19G: Proliferation assay of acalabrutinib and Compound A (combination therapy) in BTK C481S TMD8 cells (curve shift analysis).

[0109] Figure 20: Excess over Bliss score matrix of Compound A and acalabrutinib in BTK C481S TMD8 cells.

[0110] Figure 21 : Ibrutinib resistance mutations can be detected before clinical relapse. For 20 patients with a detectable mutation in BTK or PLCG2 at time of relapse, samples before relapse were analyzed retrospectively to determine the interval of time between mutation detection and clinical relapse. An initial clone could be detected at an estimated median of 9.3 months before relapse. UPN, unique patient number (Woyach et al. 2017).

[0111] Detailed description of the invention

[0112] The inventors have established that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular a macrocyclic reversible BTK inhibitor having any one of Formula (l-a) to (l-h) as defined below, is more effective than an irreversible or reversible BTK inhibitor alone.

[0113] In particular, it has now been shown that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular a compound having any one of Formula (l-a) to (l-h), as defined above, is more effective than an irreversible or reversible BTK inhibitor alone in inhibiting B-cell lymphoma cells comprising two separate cell pools each containing a different mutation in BTK. Moreover, combinations of a reversible BTK inhibitor compound according to Formula (l-a) to (l-h) and an irreversible, covalent, BTK inhibitor is synergistically effective in inhibiting B-cell lymphoma cells. In specific examples, a synergistic effect of BTK inhibition has been shown in B-cell lymphoma cell line in which BTK has a C481S mutation. In preferred embodiments, the reversible BTK inhibition compounds according to the invention have any one of Formula (l-a) to (l-h), which are extensively described in co-pending PCT application PCT / EP2021 / 085641 and in PCT application PCT / EP2022 / 085765. The reversible BTK inhibitors according to the present invention include the (macrocyclic) BTK inhibition compounds of the co-pending PCT application PCT / EP2021 / 085641 and of the PCT application PCT / EP2022 / 085765 and are incorporated by reference, including the exemplary compounds having sub-formula 1 - 226 and the described syntheses routes for manufacturing the compounds having any one of Formula (l-a) to (l-h).

[0114] Surprisingly, the inventors have found that macrocyclic reversible BTK inhibitiors according to the invention provide an improved reversible binding activity towards wild-type BTK and / or BTK mutants than what is typically obtained with reversible BTK inhibitors. The compounds according to the invention have any one of Formula (l-a) to (l-h), which contains a macrocyclic moiety, in combination with BTK specific pharmacophores (e.g. based on ligands for binding to BTK) to provide a binding activity towards wild-type BTK and / or BTK mutants through improved reversible binding. In particular it has been found that the compound provides a considerable longer residence time than what is typically obtained with reversible BTK inhibitors.

[0115] Embodiments

[0116] Reversible BTK inhibitors

[0117] In preferred embodiments the reversible BTK inhibitor is a compound according to any one of Formula (l-a) to (l-h), or pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof:

[0118] (Formula l-a) (Formula l-b) (Formula l-c) (Formula l-d)

[0119] 15

[0120] (Formula l-e) (Formula l-f) (Formula l-g) (Formula l-h)

[0121] Wherein R1is , wherein :

[0122] W is an aryl group having 6-10 carbon or a heteroaryl group having 1 -5 carbon; wherein any said aryl group and heteroaryl group is optionally and independently substituted with one or more substituents selected from halogen, (1 -2C)alkyl, (1 -2C)alkoxy; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three fluoro;

[0123] V is any one of O, -C(O)-NH-, -NH-C(O)-, -CH(R1v)-NH-C(O)-, -CH(R1v)- ;

[0124] R1vis hydrogen or (1 -2C)alkyl;

[0125] U is an aryl group having 6-10 carbon or an heteroaryl group having 1 -5 carbon; wherein any of said aryl group and heteroaryl group is optionally and independently substituted with one or more substituents selected from halogen, cyano, (1 -4C)alkyl, (1 -5C)alkoxy, (3-6C)cycloalkyl or (3-6C)heterocycloalkyl; wherein any of said alkyl, alkoxy, cycloalkyl and heterocycloalkyl group is optionally and independently substituted with one, two or three halogen; wherein R2is of Formula (ll-a) to (ll-f) selected from the group consisting of:

[0126] (Formula ll-a) (Formula ll-b) (Formula ll-c) and

[0127] (Formula ll-d) (Formula ll-e) (Formula ll-f) wherein Q is a monocyclic ring selected from a (3-7C)cycloalkyl and a (3- 6C)heterocycloalkyl, wherein Xi , X2 and X3 are independently selected from CH2, - CH2CH2-, O, N and a direct bond; wherein any of the cycloalkyl, heterocycloalkyl and alkyl group is optionally and independently substituted with one or more substituents selected from halogen, hydroxy, (1 -3C)alkyl, (1 -3C)alkoxy, (1 -4C)alkylcarbonyl or (3- 4C)cycloalkyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three halogen; wherein R3and R4together represent a linker having Formula (111-1 to III-40) selected from the group consisting of: n = 0 - 6 n = 1 - 5 n = 0 - 4 n = 0 - 4 n = 1 - 5

[0128] (III-1 ) (III-2) (III-3) (III-4) (III-5)

[0129] whereby the marks the position of R3in any one of Formula l-a to l-h, and whereby the marks the position of R4in any one of Formula Il-a to ll-f; wherein any of said linkers is optionally and independently substituted with one or more substituents selected from deuterium, halogen, oxo, hydroxy, CD3, (1 -4C)alkyl, (1 - 5C)alkoxy, (3-6C)cycloalkyl, (3-6C)cycloalkoxy and (1 -6C)alkylcarbonyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three halogen.

[0130] In more preferred embodiments the reversible BTK inhibitor is a compound according to Formula (l-a) or (l-b) or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound comprises a bicyclic scaffold selected from: and

[0131] (Formula l-a) (Formula l-b)

[0132] Wherein R1is any one of: and wherein R2wis selected from hydrogen, halogen, (1 -2C)alkyl, (1-2C)alkoxy; wherein any said alkyl or alkoxy group is optionally and independently substituted with one, two or three fluoro; wherein R3uis selected from hydrogen, halogen, cyano, (1 -4C)alkyl, (1 -5C)alkoxy, (3- 6C)cycloalkyl or (3-6C)heterocycloalkyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three fluoro; wherein R2is selected from the group consisting of:

[0133] (Formula Il-a3) (Formula Il-a4) (Formula Il-b3) (Formula Il-c3)

[0134] (Formula ll-d) (Formula ll-f) (Formula II-a5) (Formula II-b4) wherein any of said cycloalkyl, heterocycloalkyl and alkyl group is optionally and independently substituted with hydroxy, methyl, acetyl or methoxy; wherein R3and R4together represent a linker having Formula selected from the group consisting of:

[0135] 15

[0136] 20

[0137] 25 whereby the marks the position of R3in any one of Formula l-a to l-b, and whereby the marks the position of R4in any one of Formula Il-a to ll-f; wherein any of said linkers is optionally and independently substituted with one or more substituents selected from deuterium, halogen, oxo, hydroxy, CD3, (1 -4C)alkyl, (1-5C)alkoxy, (3- 6C)cycloalkyl, (3-6C)cycloalkoxy and (1 -6C)alkylcarbonyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three halogen. In particular embodiments the reversible BTK inhibitor is a compound according to Formula (A):

[0138] Formula (A), or pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

[0139] Irreversible BTK inhibitors

[0140] In preferred embodiments, the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, spebrutinib, branebrutinib, evobrutinib, remibrutinib, tolebrutinib, orelabrutinib, elsubrutinib, edralbrutinib, ACP-5862, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

[0141] In a preferred embodiment, the irreversible BTK inhibitor of the combination is ibrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

[0142] In a preferred embodiment, the irreversible BTK inhibitor of the combination is acalabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

[0143] In a preferred embodiment, the irreversible BTK inhibitor of the combination is zanubrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

[0144] In a preferred embodiment, the irreversible BTK inhibitor of the combination is tirabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

[0145] BTK Protein Sequence

[0146] SEQ ID NO: 1 tyrosine-protein kinase BTK [Homo sapiens]

[0147] Mutant BTK

[0148] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is C481S.

[0149] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of cysteine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 481 of the BTK protein.

[0150] In a more preferred embodiment, the modification is a substitution of cysteine to serine at amino acid position 481 of the BTK protein.

[0151] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 416 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is V416L.

[0152] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 416 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of valine to an amino acid selected from leucine, isoleucine, cysteine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 416 of the BTK protein.

[0153] In a more preferred embodiment, the modification is a substitution of valine to leucine at amino acid position 416 of the BTK protein.

[0154] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 428 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is A428D.

[0155] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 428 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of alanine to an amino acid selected from leucine, isoleucine, valine, cysteine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 428 of the BTK protein.

[0156] In a more preferred embodiment, the modification is a substitution of alanine to aspartic acid at amino acid position 428 of the BTK protein.

[0157] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 437 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is M437R.

[0158] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 437 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of methionine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, cysteine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 437 of the BTK protein.

[0159] In a more preferred embodiment, the modification is a substitution of methionine to arginine at amino acid position 437 of the BTK protein.

[0160] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 474 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is T474I. In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 474 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of threonine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, methionine, serine, cysteine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 474 of the BTK protein.

[0161] In a more preferred embodiment, the modification is a substitution of threonine to isoleucine at amino acid position 474 of the BTK protein.

[0162] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 528 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is L528W.

[0163] In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 528 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of leucine to an amino acid selected from cysteine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 528 of the BTK protein.

[0164] In a more preferred embodiment, the modification is a substitution of leucine to tryptophane at amino acid position 528 of the BTK protein.

[0165] Prior therapy

[0166] In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, ibrutinib before starting a new treatment option for managing the same condition.

[0167] In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, acalabrutinib before starting a new treatment option for managing the same condition.

[0168] In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, zanubrutinib before starting a new treatment option for managing the same condition.

[0169] In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, tirabrutinib before starting a new treatment option for managing the same condition.

[0170] Maintenance Therapy

[0171] In preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, ibrutinib.

[0172] In preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, acalabrutinib.ln preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, zanubrutinib.

[0173] In preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, tirabrutinib.

[0174] Provided herein are embodiments of methods including maintenance therapy of subject having a B-cell hematological malignancy. In some embodiments, the methods for maintenance therapy comprise treating a B-cell hematological malignancy with a covalent and / or irreversible BTK inhibitor for an initial treatment period, followed by a maintenance therapy regimen. In some embodiments, the methods for maintenance therapy comprise treating a B-cell haematological malignancy with a covalent and / or irreversible BTK inhibitor for a period of six months or longer, such as, for example, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or longer.

[0175] Monitoring subject

[0176] In preferred embodiments, the subject is monitored over the course of the therapy with a BTK inhibitor for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor.

[0177] In preferred embodiments, the subject is monitored at predetermined intervals of time over the course of the therapy for treatment of the hyperproliferative disease, wherein the predetermined interval of time is every week, every 2 weeks, every 3 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 1 1 months, every year following the first administration of the covalent and / or irreversible BTK inhibitor. The treatment which is monitored is in particular a treatment of administration of an irreversible BTK inhibitor.

[0178] Methods for monitoring subject

[0179] In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 481 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 481 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 481 of the BTK protein.

[0180] In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 481 ; and (b) does not bind to nucleic acids encoding the wild-type BTK having cysteine at amino acid position 481 . In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

[0181] In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 416 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 416 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 416 of the BTK protein.

[0182] In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 416; and (b) does not bind to nucleic acids encoding the wild-type BTK having valine at amino acid position 416. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

[0183] In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 428 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 428 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 428 of the BTK protein.

[0184] In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 428; and (b) does not bind to nucleic acids encoding the wild-type BTK having alanine at amino acid position 428. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

[0185] In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 437 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 437 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 437 of the BTK protein.

[0186] In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 437; and (b) does not bind to nucleic acids encoding the wild-type BTK having methionine at amino acid position 437. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

[0187] In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 474 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 474 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 474 of the BTK protein.

[0188] In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 474; and (b) does not bind to nucleic acids encoding the wild-type BTK having threonine at amino acid position 474. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

[0189] In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 528 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 528 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 528 of the BTK protein.

[0190] In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 528; and (b) does not bind to nucleic acids encoding the wild-type BTK having tryptophane at amino acid position 528. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

[0191] In some embodiments, the sample for use in the methods contains one or more tumor cells from the subject. In some embodiments, the sample for use in the methods contains cell free DNA (cfDNA). In some embodiments, the sample for use in the methods contains circulating tumor DNA (ctDNA). In some embodiments, the sample for use in the methods contains tumor derived extracellular vesicles (EV). In some embodiments, the sample is a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate.

[0192] In some embodiments of the methods, the nucleic acids used in the method is isolated from a tumor cell sample from the subject. In some embodiments of the methods, the nucleic acids used in the method are isolated from extracellular vesicles (EV) isolated from the subject. In some embodiments of the method the nucleic acids used in the method are isolated from a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate from the subject. In some embodiments of the methods, the nucleic acids for use in the assay are RNA or DNA. In some embodiments of the methods, the nucleic acids for use in the assay are genomic DNA. In some embodiments of the methods, the nucleic acids for use in the assay are total RNA. In some embodiments of the methods, the nucleic acids for use in the assay are messenger RNA (mRNA).

[0193] In some embodiments of the methods, the nucleic acids for use in the assay are complementary DNA (cDNA).

[0194] In some embodiments of the methods, the method further comprises isolating mRNA from the RNA sample. In some embodiments of the methods, the method further comprises reverse transcription of the total RNA, or mRNA, into cDNA.

[0195] In preferred embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 481 ; and (b) does not bind to nucleic acids encoding the wild-type BTK having cysteine at amino acid position 481 . In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

[0196] In preferred embodiments, the sample for use in the methods contains one or more tumor cells from the subject. In preferred embodiments, the sample for use in the methods contains circulating tumor DNA (ctDNA). In preferred embodiments, the sample is a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate.

[0197] In preferred embodiments of the methods, the nucleic acids used in the method is isolated from a tumor cell sample from the subject. In preferred embodiments of the method the nucleic acids used in the method are isolated from a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate from the subject.

[0198] In preferred embodiments of the methods, the nucleic acids for use in the assay is genomic DNA.

[0199] Definition subject - recurrent or refractory form of a hyperproliferative disease

[0200] In preferred embodiments, the subject is diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, which subject has previously been treated with an irreversible BTK inhibitor.

[0201] At risk of a recurrent or refractory form of a hyperproliferative disease in the context of this invention is defined as the subject having a mutation in a gene that results in expression of a mutated BTK protein that is unable to bind a BTK inhibitor, preferably an irreversible BTK inhibitor.

[0202] In preferred embodiments, the subject is diagnosed with recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy that is resistant, or non-responsive to therapy with an irreversible BTK inhibitor.

[0203] In preferred embodiments, the subject is identified as having a mutation in a gene that results in expression of a mutated BTK protein that is unable to bind a BTK inhibitor, preferably an irreversible BTK inhibitor, which provides a risk of a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy.

[0204] Definition relapse or refractory of disease

[0205] In preferred embodiments, the combination therapy is started when the B-cell hematological malignancy of the subject is relapsed or refractory, preferably wherein CLL / SLL is relapsed or refractory.

[0206] In preferred embodiments, the combination therapy is started when the B-cell hematological malignancy of the subject is relapsed or refractory and the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 , of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the mutant modification is C481S.

[0207] Combination therapy

[0208] Combination - selection irreversible inhibitor

[0209] In preferred embodiments, the irreversible BTK inhibitor of the combination therapy is the same as the irreversible BTK inhibitor of a prior (maintenance) therapy. In a particular example, the irreversible BTK inhibitor of the combination therapy is ibrutinib, while the irreversible BTK inhibitor of a prior (maintenance) therapy is also ibrutinib.

[0210] In alternative embodiments, the irreversible BTK inhibitor of the combination therapy is different from the irreversible BTK inhibitor of a prior (maintenance) therapy. In a particular example, the irreversible BTK inhibitor of the combination therapy is acalabrutinib, while the irreversible BTK inhibitor of a prior (maintenance) therapy is ibrutinib.

[0211] Dose - therapeutically combinatory amount

[0212] The term "therapeutically combinatory amount” in preferred embodiments, is the total amount of the two (or more) compounds selected of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular the total amount of the irreversible BTK inhibitor and the macrocyclic reversible BTK inhibitor, used for treating the hyperproliferative disease, in particular a B-cell hematological malignancy. In preferred embodiments, the irreversible BTK inhibitor of the combination is administered in an amount in the range of 70 - 750 mg / day and / or wherein the irreversible BTK inhibitor of the combination is administered using one or more unit doses having an amount in the range of 70 - 750 mg / unit dose.

[0213] In preferred embodiments, the irreversible BTK inhibitor of the combination is administered daily at a dose selected from the group consisting of 70 mg, 100 mg, 140 mg, 160 mg, 200 mg, 280 mg, 320 mg, 420 mg, 480 mg, and 560 mg per day and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

[0214] In preferred embodiments, the irreversible BTK inhibitor of the combination is administered twice daily using a unit dose of 80 mg, 100 mg or 160 mg and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

[0215] In preferred embodiments, the dose amount of the irreversible BTK inhibitor of the therapeutically combinatory amount of the combination is lower than the dose amount of the same irreversible BTK inhibitor administered during the prior therapy.

[0216] In preferred embodiments, the therapeutically combinatory amount of the dose of the irreversible BTK inhibitor and the dose of the reversible BTK inhibitor of the combination is lower than the dose of the irreversible BTK inhibitor, which was administered during the prior therapy.

[0217] Dosage forms

[0218] In preferred embodiments, the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in a combined dosage form.

[0219] In alternative preferred embodiments, the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in separate dosage forms.

[0220] Pharmaceutical composition

[0221] In a preferred embodiment, the pharmaceutical composition comprises a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, wherein the reversible BTK inhibitor is a compound according to any one of Formula (l-a) to (l-h), in a particular example a compound according to Formula A.

[0222] Pharmaceutical compositions in accordance with the present invention may comprise, as one of the active ingredients (‘API’), compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0223] As used herein, “a pharmaceutically acceptable salt” includes any salt that retains the activity of the active agent(s) and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. Preferably, the pharmaceutically acceptable salt is the HCI-salt of the compound of the invention. The pharmaceutically acceptable salt of the disclosed compounds may be prepared by methods of pharmacy well known to those skilled in the art.

[0224] Furthermore, the compositions can comprise compounds according to the invention in the form of a solvate, comprising a pharmaceutically acceptable solvent, such as water (‘hydrate’), ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

[0225] As used herein, the term “pharmaceutical composition” refers to a composition comprising a compound according to the invention or a salt or solvate thereof and, as the case may be, one or more additional, non-toxic ingredients, which composition is in a form suitable for administration to a (human) subject, through any route of administration, and which composition is physiologically tolerated upon such administration.

[0226] The compositions of the invention may thus comprise one or more additional ingredients. In a preferred embodiment, the composition comprises one or more carriers and / or excipients. As is known by those of average skill in the art, the appropriate choice of excipients is dependent on multiple factors, including the physicochemical properties of the API, the preferred pharmaceutical form, the preferred route of administration, the desired rate of release, etc. The compositions of the invention can be formulated for a variety of routes of administration, oral administration being particularly preferred. It is within the purview of those of average skill in the art to conceive and develop suitable formulations, relying on the common general knowledge as reflected in text books such as Remington’s Pharmaceutical Sciences (Meade Publishing Co., Easton, Pa., 20.sup.th Ed., 2000), the entire disclosure of which is herein incorporated by reference, and routine development efforts.

[0227] In accordance with the various aspects of the invention, the composition is preferably provided in a unit dosage form. The term ‘unit dosage form’ refers to a physically discrete unit suitable as a unitary dosage for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with any suitable pharmaceutical carrier(s) and / or excipient(s). Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gelcap as well as any metered volume of a solution, suspension, syrup or elixir or the like, which may be contained, for instance in a vial, syringe, applicator device, sachet, spray, micropump etc. In accordance with particularly preferred embodiments of the invention, the unit dosage form, is a unit dosage form that is suitable for oral administration. Most preferably, it is a solid unit dosage form, such as a tablet. Besides the compound according to the invention as such, pharmaceutically acceptable salts thereof may also be used. Pharmaceutically acceptable salts of compounds of the invention include the acid addition and base salts thereof, such as preferably the calcium, potassium or sodium salts. For a review on suitable salts, reference is made “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0228] Pharmaceutically acceptable salts of compounds according to the invention may be readily prepared by mixing together solutions of compounds according to the invention and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised.

[0229] Medical Use

[0230] The compounds and the pharmaceutical compositions of the present invention are useful as inhibitors of tyrosine kinases, in particular of BTK. In particular, compounds of this invention are useful as inhibitors of tyrosine kinases that are important in hyper-proliferative diseases, especially in cancer, such as a B-cell hematological malignancy, and in the process of angiogenesis.

[0231] The reversible BTK inhibitor compounds according to the invention having Formula (l-a) to (l-h) and pharmaceutical compositions comprising these, either alone or in combination with an irreversible BTK inhibitor, thereof can be used to treat or prevent a variety of conditions, diseases or disorders mediated by Bruton’s Tyrosine kinase (BTK).

[0232] Such conditions, diseases or disorders include: cancers or tumors, including alimentary / gastrointestinal tract cancer, colon cancer, liver cancer, skin cancer including mast cell tumor and squamous cell carcinoma, breast and mammary cancer, ovarian cancer, prostate cancer, B-cell hematological malignancy, lymphoma and leukemia (including but not limited to acute myelogenous leukemia, chronic myelogenous leukemia, mantle cell lymphoma, NHL B cell lymphomas (e.g. precursor B-ALL, marginal zone B-cell lymphoma, chronic lymphocytic leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, mediastinal large B-cell lymphoma), Hodgkin lymphoma, NK and T cell lymphomas, TEL-Syk and ITK-Syk fusion driven tumors, myelomas including multiple myeloma, myeloproliferative disorders kidney cancer, lung cancer, muscle cancer, bone cancer, bladder cancer, brain cancer, melanoma including oral and metastatic melanoma, Kaposi’s sarcoma, proliferative diabetic retinopathy, and angiogenic- associated disorders including solid tumors, and pancreatic cancer.

[0233] In preferred embodiments, the hyperproliferative disease is a B-cell hematological malignancy. In particular embodiments, the B-cell hematological malignancy is any one of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCB DLBCL), primary mediastinal B-cell lymphoma (PMBL), non- Hodgkin lymphoma, Burkitt’s lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia, lym- phoplasmacytic lymphoma / Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0234] In preferred embodiments, the B-cell hematological malignancy is mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large 13- cell lymphoma (DLBCL), Waldenstrom macroglobulinemia (WM), follicular lymphoma (FL) and marginal zone lymphoma (MZL).

[0235] In preferred embodiments, the B-cell malignancy is Mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL).

[0236] Composition / Formulation

[0237] The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0238] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0239] For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0240] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

[0241] Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0242] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0243] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

[0244] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0245] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or aa nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds can be formulated for parenteral administration by injection, e.g. bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0246] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0247] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0248] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0249] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0250] An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w / v benzyl alcohol, 8% w / v of the nonpolar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co- solvent system (VPD:5W) consists of VPD diluted 1 :1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0251] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0252] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0253] Many of the compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.

[0254] Synthesis of compounds

[0255] The compounds of the present invention can be prepared by methods well known in the art of organic chemistry. See, for example, J. March, ‘Advanced Organic Chemistry' 4thEdition, John Wiley and Sons. During synthetic sequences it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This is achieved by means of conventional protecting groups, such as those described in T.W. Greene and P.G.M. Wutts ‘Protective Groups in Organic Synthesis’ 3rdEdition, John Wiley and Sons, 1999. The protective groups are optionally removed at a convenient subsequent stage using methods well known in the art.

[0256] The products of the reactions are optionally isolated and purified, if desired, using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography and the like. Such materials are optionally characterized using conventional means, including physical constants and spectral data.

[0257] Compounds of any one of Formula l-a to l-h, wherein R1to R4and W, V and U have the previously defined meanings, can be prepared by the general synthetic route shown in any one of scheme I - XII, as described in co-pending PCT application PCT / EP2021 / 085641 and of the PCT application PCT / EP2022 / 085765.

[0258] Examples of synthesis of compounds

[0259] Intermediate 1

[0260] Benzyl (1 R,5R)-5-hvdroxycyclohex-3-ene-1 -carboxylate

[0261] (a) (1 R,4R,5R)-4-iodo-6-oxabicyclo[3.2.1 ]octan-7-one

[0262] (R)-(+)-3-Cyclohexenecarboxylic acid (50.7 g, 402 mmol) was suspended in H2O (400 mL) under nitrogen. The mixture was cooled to 4 °C and sodium bicarbonate (101 .3 g, 1 .21 mol) was added, followed by a solution of potassium iodide (333 g, 2.01 mol) and iodine (107 g, 422 mmol) in H2O (400 mL). The reaction was allowed to come to room temperature and stirred o / n and then extracted with dichloromethane (4x100 mL). The combined organic layers were washed with sat. NaHSO3-solution (2x50 mL). The organic layer was protected from light, dried over Na2SO4, filtered and concentrated (20 mbar) to afford (1 R,4R,5R)-4-iodo-6- oxabicyclo[3.2.1 ]octan-7-one (90.1 g, 89.0 %) as an off-white solid.

[0263] (b) (1 R,5R)-6-oxabicyclo[3.2.1 ]oct-3-en-7-one

[0264] (1 R,4R,5R)-4-iodo-6-oxabicyclo[3.2.1 ]octan-7-one (90.1 g, 358 mmol) was dissolved in dry THF (650 mL). Then, DBU (77 mL, 515 mmol) was added and the mixture was refluxed for 6 h. After cooling to room temperature, the suspension was filtered through Celite™, and concentrated in vacuo to -250 mL. This was used directly in the next step.

[0265] (c) Benzyl (1 R,5R)-5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 1)

[0266] To the THF solution of (1 R,5R)-6-oxabicyclo[3.2.1 ]oct-3-en-7-one (0.4 mol) in methanol (300 mL) was added 2M NaOH-solution (300 mL) and the mixture was stirred for 15 min at room temperature. The reaction was quenched by addition of 3M HCI-solution (300 mL) and the water layer was saturated by addition of sodium chloride. The mix]ure was extracted with ethyl acetate (3x100 mL). The combined organic phases were washed with brine, dried over sodium sulfate, filtered and the solvent removed in vacuo. The residue was dissolved in DMF (800 ml), cesium carbonate (129 g, 0.4 mol) and benzyl bromide (57 mL, 0.48 mol) were added subsequently. The mixture was stirred at room temperature for 30 min. The precipitate formed was filtered and the precipitate was washed with diethylether. The filtrate was washed with water, brine, dried over sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography (heptane / ethyl acetate = 95 / 5 to 45 / 55 v / v%) to give benzyl (1 R,5R)-5- hydroxycyclohex-3-ene-1 -carboxylate (57.1 g, 61 .5% over 3 steps) as a cream-coloured oil.

[0267] Intermediate 2

[0268] Ethyl (1 R,5R)-5-hvdroxycyclohex-3-ene-1 -carboxylate

[0269] (a) (1 R,4R,5R)-4-iodo-6-oxabicyclo[3.2.1 ]octan-7-one

[0270] (R)-(+)-3-Cyclohexenecarboxylic acid (50.7 g, 0.4 mol) was suspended in H2O (400 mL) under nitrogen. The reaction mixture was cooled to 4 °C and sodium bicarbonate (101 g, 1 .2 mol) was added, followed by a solution of potassium iodide (333 g, 2 mol) and iodine (107 g, 0.42 mol) in H2O (400 mL). The reaction was allowed to come to room temperature and stirred o / n and then extracted with DCM (4x150 mL). The combined organic layers were washed with a solution of Na2S2O3(120 g) in H2O (600 mL). The aqueous layer was extracted with DCM (2x150 mL). The combined organic layers were protected from light, dried over Na2SO4, filtered and concentrated (20 mbar) to afford (1 R,4R,5R)-4-iodo-6-oxabicyclo[3.2.1 ]octan-7-one (95.22 g, 94.5 %) as an off-white solid.

[0271] (b) (1 R,5R)-6-oxabicyclo[3.2.1 ]oct-3-en-7-one

[0272] (1 R,4R,5R)-4-iodo-6-oxabicyclo[3.2.1 ]octan-7-one (95.22 g, 377.9 mmol) was dissolved in dry THE (700 mL). Then, DBU (86.3 g, 566.9 mmol) was added and the mixture was refluxed o / n. The reaction mixture was cooled to room temperature, diluted with diethylether (500 mL) and extracted with aq. HCI (1 L, 1 M) and brine (250 mL). The aqueous layers were extracted with diethylether (2 x 480 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated (350 mbar to afford (1 R,5R)-6-oxabicyclo[3.2.1 ]oct-3-en-7-one quantitatively as a yellowish oil which was used directly in the next step.

[0273] (c) Ethyl (1 R,5R)-5-hvdroxycyclohex-3-ene-1 -carboxylate (Intermediate 2)

[0274] To a stirred solution of (1 R,5R)-6-oxabicyclo[3.2.1 ]oct-3-en-7-one (377.9 mmol, theor.) in ethanol (750 mL) was added potassium carbonate (10.45 g, 75.6 mmol) at room temperature and the mixture stirred o / n. The reaction mixture was filtered through a Celite pad. Removal of ethanol under reduced pressure afforded the crude product that was purified by column chromatography plug filtration (eluent 40% EtOAc / heptane) to afford the title compound (41 .38 g, 60.8% over 3 steps and column) as a yellow liquid. Intermediate 3

[0275] Methyl (1 R,5R)-5-hvdroxycyclohex-3-ene-1 -carboxylate

[0276] (a) (1 R,4R,5R)-4-iodo-6-oxabicyclo[3.2.11octan-7-one

[0277] (R)-(+)-3-Cyclohexenecarboxylic acid (20.2 g, 160 mmol) was suspended in H2O (430 mL) under nitrogen. The reaction mixture was cooled to 0 °C and sodium bicarbonate (40.3 g, 480.3 mmol) was added, followed by a solution of potassium iodide (159.5 g, 961 mmol) and iodine (39.6 g, 168 mmol) in H2O (360 mL). The reaction was allowed to come to room temperature and stirred o / n and then extracted with DCM (3x150 mL). The combined organic layers were washed with a solution of Na2S2O3(120 g) in H2O (600 mL). The aqueous layer was extracted with DCM (2x150 mL). The combined organic layers were protected from light, dried over Na2SO4, filtered and concentrated (20 mbar) to afford (1 R,4R,5R)4-iodo-6- oxabicyclo[3.2.1 ]octan-7-one (37.88 g, 93.9 %) as an off-white solid.

[0278] (b) (1 R,5R)-6-oxabicyclo[3.2.1 ]oct-3-en-7-one

[0279] (1 R,4R,5R)-4-iodo-6-oxabicyclo[3.2.1 ]octan-7-one (37.88 g, 150.3 mmol) was dissolved in dry THF (750 mL). Then, DBU (34.3 g, 225.2 mmol) was added and the mixture was refluxed o / n. The reaction mixture was cooled to room temperature, diluted with diethylether (480 mL) and extracted with aq. HCI (1 L, 0.5 M) and brine (1 L). The aqueous layers were extracted with diethylether (2 x 480 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated (350 mbar to afford (1 R,5R)-6-oxabicyclo[3.2.1 ]oct-3-en-7-one quantitatively as a yellowish oil which was used directly in the next step.

[0280] (c) Methyl (1 R,5R)-5-hvdroxycyclohex-3-ene-1 -carboxylate (Intermediate 3)

[0281] Sodium bicarbonate (37.88 g, 0.451 mol) was added to a solution (1 R,5R)-6- oxabicyclo[3.2.1 ]oct-3-en-7-one (150.3 mmol theor.) in anhydrous MeOH (300 mL). After stirring for 1 week at room temperature the solvent was removed in vacuo (40 °C / 300 mbar). The residue was diluted with water (500 mL) and extracted with dichloromethane (3x250 mL). The combined extracts were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (21 .2 g, 90.3%) as a slightly coloured liquid. Intermediate 4 (Route A)

[0282] (1 R,3R)-3-(Benzyloxycarbonylamino)cyclohexanecarboxylic acid

[0283] (a) Methyl (1 R,5S)-5-[bis(tert-butoxycarbonyl)amino1cyclohex-3-ene-1 -carboxylate

[0284] To an ice-cold (4 °C) solution of di-tert-butyl iminodicarboxylate (4.6 g, 21 .2 mmol), methyl (1 R,5R)-5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 3) (3.31 g, 21 .2 mmol) and triphenylphosphine (6.67 g, 25.4 mmol) in 2-MeTHF (180 mL) was added dropwise a solution of diisopropyl azodicarboxylate (6.26 mL, 31 .8 mmol) in 2-MeTHF (30 mL). The mixture was stirred for 30 min at 4 °C and then allowed to warm to room temperature and stirred for 3 h. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography using SiO2and heptane / ethyl acetate = 3 / 1 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 5.68 g of methyl (1 R,5S)-5- [bis(tert-butoxycarbonyl)amino]cyclohex-3-ene-1 -carboxylate (yield 75.4 %).

[0285] (b) Methyl (1 R,3R)-3-[bis(tert-butoxycarbonyl)amino1cyclohexanecarboxylate

[0286] To a solution of methyl (1 R,5S)-5-[bis(tert-butoxycarbonyl)amino]cyclohex-3-ene-1 - carboxylate (2.48 g, 6.98 mmol) in ethanol (140 mL) was added 248 mg of 10% Pd / C. Catalytic hydrogenation was performed at room temperature for 16 h. The palladium-catalyst was filtered and the filtrate was concentrated in vacuo to give 2.6 g of the title compound (Yield: quantitative).

[0287] (c) Methyl (1 R,3R)-3-aminocyclohexanecarboxylate hydrochloride

[0288] To methyl (1 R,3R)-3-[bis(tert-butoxycarbonyl)amino]cyclohexanecarboxylate (2.6 g, 7.2 mmol) was added 4M HCI / dioxane solution (18 mL) and the mixture was stirred at room temperature o / n. The mixture was concentrated in vacuo to give 1 .06 g the title compound (yield: 76%).

[0289] (d) Methyl (1 R,3R)-3-(benzyloxycarbonylamino)cyclohexanecarboxylate

[0290] Methyl (1 R,3R)-3-aminocyclohexanecarboxylate hydrochloride (1 .06 g, 5.47 mmol) was suspended in 10 mL water. Sodium bicarbonate (1 .38 g, 16.4 mmol) in 10 mL water was added followed by a drop-wise addition of a solution N-(benzyloxycarbonyloxy)succinimide (1 .50 g, 6.01 mmol) in dioxane (30 mL). The reaction mixture was stirred at room temperature o / n. The mixture was diluted with ethyl acetate (50 mL) and water (50 mL) and the bi-phasic system was stirred 30 minutes at room temperature. The layers were separated and the water layer was extracted with ethyl acetate (2x20 mL). The combined organic layers were washed with water (50 mL), 0.5N aq. HCI-solution (50 mL), water (50 mL), 5% aq. NaHSO3-solution (50 mL), water (50 mL) and brine (25 mL), dried ( Na2SO4), filtered and concentrated in vacuo to give 1 .78 g of the title compound (yield: quantitative, crude).

[0291] (e) (1 R,3R)-3-(Benzyloxycarbonylamino)cyclohexanecarboxylic acid (Intermediate 4)

[0292] The crude product methyl (1 R,3R)-3-(benzyloxycarbonylamino)cyclohexanecarboxy- late (1 .45 g, 4.98 mmol) was dissolved in THF / dioxane / water = 4 / 1 / 1 v / v% (74 mL) and subsequently lithium hydroxide (358 mg, 14.9 mmol) was added. The mixture was stirred at room temperature o / n. Ethyl acetate (50 mL) and water (were added) and the pH of the mixture was adjusted to pH < 3 by addition of 2M HCI-solution. The organic phase was separated, washed with water, brine, dried over sodium sulfate , filtered and concentrated under reduced pressure to give 800 mg of (1 R,3R)-3-(benzyloxycarbonylamino)cyclohexanecarboxylic acid (Intermediate 4) (yield:57.9%).

[0293] Intermediate 4 (Route B)

[0294] (1 R,3R)-3-(Benzyloxycarbonylamino)cyclohexanecarboxylic acid

[0295] (a) tert-Butyl N-(4-nitrophenyl)sulfonylcarbamate

[0296] Triethylamine (10.4 mL, 74.62 mmol), 4-dimethylaminopyridine (605 mg, 4.95 mmol) and di-tert-butyl dicarbonate (13.5 g, 61 .86 mmol) were added sequentially to a solution of 4- nitrobenzene sulfonamide (10 g, 49.46 mmol) in dichloromethane (100 mL). The reaction mixture was stirred for 30 minutes at room temperature. To the reaction mixture was added hydrochloric acid (1 N aqueous solution) until it becomes acidic. The organic layer was separated and washed with saturated sodium chloride aqueous solution, dried over sodium sulfate, filtered and then concentrated under reduced pressure. The residue was dissolved in dichloromethane and purified by column chormatography over silica (heptane to ethyl acetate = 10 / 0 to 0 / 10) to give 13.09 g of the title compound (yield: 87.5%).

[0297] (b) Methyl (1 R,5S)-5-[tert-butoxycarbonyl-(4-nitrophenyl)sulfonyl-amino1-cyclohex-3-ene-1 - carboxylate

[0298] To a cold (-20 °C) solution of methyl (1 R,5R)-5-hydroxycyclohex-3-ene-1 -carboxylate (15 g, 96.0 mmol), tert-butyl N-(4-nitrophenyl)sulfonylcarbamate (29.0 g, 96.0 mmol) and triphenylphosphine (27.7 g, 105.6 mmol) in THF (300 mL) was added dropwise a solution of diisopropyl azodicarboxylate (20.8 mL, 105.6 mmol) in THF (100 mL). The reaction mixture was concentrated under reduced pressure to give a residue which was purified by column chromatography (hexane / ethyl acetate = 85 / 15 v / v%) to give 36 g of the title compound, (yield: 85.1 %).

[0299] (c) Methyl (1 R,5S)-5-(tert-butoxycarbonylamino)cyclohex-3-ene-1 -carboxylate

[0300] To a stirred solution of methyl (1 R,5S)-5-[tert-butoxycarbonyl-(4-nitrophenyl)sulfonyl- amino]-cyclohex-3-ene-1 -carboxylate (35.15 g, 79.8 mmol) in acetone (300 mL) was added DBU (23.85 mL, 159.6 mmol) and 2-mercaptoethanol ( 11.23 mL, 159.6 mmol). The reaction mixture was stirred for 3 h at room temperature. Acetone was removed under reduced pressure and the resulting residue was purified by column chromatography (hexane / ethyl acetate = 90 / 10 to 88 / 12 v / v%) to give 14.1 g of the title compound (Yield: 69.2%) as a crystalline white solid.

[0301] (d) Methyl (1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylate

[0302] To a solution of methyl (1 R,5S)-5-(tert-butoxycarbonylamino)cyclohex-3-ene-1 - carboxylate (14.9 g, 58.36 mmol) in methanol (300 mL) was added 1 .5 g of 10% Pd / C. Catalytic hydrogenation was performed for 3 h at room temperature. The palladium-catalyst was filtered and the filtrate was concentrated in vacuo to afford the title compound in quantitative crude yield.

[0303] (e) Methyl (1 R,3R)-3-aminocyclohexanecarboxylate hydrochloride

[0304] To a cooled (4 °C) solution of methyl (1 R,3R)-3-(tert-butoxycarbonylamino)cyclo- hexanecarboxylate (15.2 g, 58.36 mmol) in methanol (300 mL) was added drop-wise acetyl chloride (42 mL, 583.6 mmol). The reaction mixture was stirred for 1 h. The mixture was concentrated under reduced pressure and dried in vacuo to give the title compound in quantitative crude yield.

[0305] (f) Methyl (1 R,3R)-3-(benzyloxycarbonylamino)cyclohexanecarboxylate

[0306] To a cooled (4 °C) solution of methyl (1 R,3R)-3-aminocyclohexanecarboxylate hydrochloride (58.36 mmol) in dioxane / water = 1 / 1 v / v% (200 mL) was added portion-wise sodium bicarbonate (14.7 g, 175 mmol). To the resulting suspension was added drop-wise a solution of N-(benzyloxycarbonyloxy)succinimide (14.8 g, 59.02 mmol) in dioxane (150 mL) and the resulting mixture was stirred at room temperature o / w. LC-MS showed some starting material present. Additionally, 1 .5 g of Z-ONSu was added as a solution in dioxane. The mixture was stirred at room temperature o / n. Ethyl acetate was added and the resulting mixture was washed with 0.5M HCI solution, water and brine. The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure and dried in vacuo to give the title compound in quantitative crude yield.

[0307] (g) (1 R,3R)-3-(benzyloxycarbonylamino)cyclohexanecarboxylic acid (Intermediate 4)

[0308] To a solution of methyl (1 R,3R)-3-(benzyloxycarbonylamino)cyclohexanecarboxylate (58.36 mmol) in THF / water = 4 / 1 v / v% (375 mL) was added lithium hydroxide (4.21 g, 175 mmol) and the reaction mixture was stirred at room temperature o / n. Ethyl acetate (300 mL) and water (300 mL) were added to the mixture and the aqueous phase was separated. The ethyl acetate layer washed extracted with water (100 mL). The combined aqueous phases were washed with dichloromethane (100 mL) and acidified (pH < 2) by addition of 2M HCI-solution (90 mL). The water layer was extracted with ethyl acetate (3x250 mL). The combined ethyl acetate layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure and dried in vacuo to give 15.72 g of the title compound (Yield: 96.7% over 4 steps).

[0309] Intermediate 5

[0310] (1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid

[0311] (a) Ethyl (1 R,5S)-5-(1 ,3-dioxoisoindolin-2-yl)cyclohex-3-ene-1 -carboxylate

[0312] To an ice-cold (0 °C) solution of ethyl (1 R,5R)-5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 2, 15.0 g, 88.13 mmol), phthalimide (14.26 g, 96.94 mmol) and triphenylphos- phine (34.67 g, 132.2 mmol) in toluene (264 mL) was added dropwise diisopropyl azodicarbo- xylate (26.02 mL, 132.2 mmol) in 10 min. The reaction mixture was stirred at 0 °C for 30 min and then allowed to come to room temperature and stirred for 3 h. The mixture was evaporated under reduced pressure to give a yellow oil. Heptane / ethyl acetate = 7 / 3 v / v% (500 mL) was added and the mixture was heated to 70 °C. After cooling, the mixture was stirred for 72 h at room temperature. The solids were filtered, washed with heptane / ethyl acetate = 9 / 1 v / v% (2x50 mL) and the filtrate was evaporated under reduced pressure. The resulting residue was purified by column chromatography (heptane / ethyl acetate = 9 / 1 to 6 / 4 v / v%) to give 21 .96 g of the title compound (Yield: 83.0%) as an off-white solid.

[0313] (b) Ethyl (1 R,3R)-3-(1 ,3-dioxoisoindolin-2-yl)cyclohexanecarboxylate

[0314] To a solution of ethyl (1 R,5S)-5-(1 ,3-dioxoisoindolin-2-yl)cyclohex-3-ene-1 -carboxylate (27.73 g, 97.19 mmol) in methanol (975 mL) was added 2.7 g of 10% Pd / C. Catalytic hydrogenation was performed for 3 h at room temperature. The palladium-catalyst was filtered and the filtrate was concentrated in vacuo to afford 27.52 g the title compound (Yield: 94%).

[0315] (c) Ethyl (1 R,3R)-3-aminocyclohexanecarboxylate

[0316] To a solution of ethyl (1 R,3R)-3-(1 ,3-dioxoisoindolin-2-yl)cyclohexanecarboxylate (26.5 g, 87.95 mmol) in ethanol (440 mL) was added drop-wise hydrazine hydrate (64% in water, 4.68 mL, 96.74 mmol). The reaction mixture was stirred for 30 min. at room temperature and then refluxed for 3 h. Additional hydrazine hydrate (425 μL) was added and stirring under reflux was continued for 2 h. The mixture was concentrated under reduced pressure and dried in vacuo to give the title compound in quantitative crude yield.

[0317] (d) Ethyl (1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylate

[0318] To a cold (0 °C) stirred suspension of ethyl (1 R,3R)-3-aminocyclohexanecarboxylate (91 .27mmol, theoretical) in dichloromethane (456ml) was added portion-wise di-tert-butyl dicarbonate (21 .91 g, 100.4 mmol). The reaction mixture was stirred for 15 min at 0 °C, then allowed to come to room temperature. The mixture was washed with cold 0.5N NaOH-solution, water and brine. The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography (heptane / ethyl acetate = 8 / 2 to 6 / 4 v / v%) to give 20.5 g of the title compound (Yield: 83.0% over two steps) as an off-white solid.

[0319] (e) (1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid

[0320] To a solution of ethyl (1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarboxylate (20.5 g, 75.56 mmol) in THE (300 mL) was added a solution of lithium hydroxide (1 .8 g, 75.56 mmol) in water (150 mL) and the reaction mixture was stirred at room temperature o / n. Additional lithium hydroxide (0.9 g) was added and stirring was continued for 24 h at room temperature. The mixture was acidified (pH < 2) by addition of 1 M HCI-solution (131 mL). The water layer was separated and extracted with dichloromethane (2x100 mL). The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure and dried in vacuo to give 16.81 g (91 %) of (1 R,3R)-3-(tert-butoxycarbonylamino)cyclo- hexanecarboxylic acid (Intermediate 5).

[0321] Intermediate 6 (6-Bromo-3-chloro-pyrazin-2-yl)methanamine hydrochloride

[0322] (a) 3-Amino-6-bromo-pyrazine-2-carbonitrile

[0323] To a solution of commercially available 2-amino-3,5-dibromo pyrazine (25.12 g, 99.3 mmol) in DMF (160 mL) was added sodium cyanide (4.97 g, 101 .3 mmol) and copper(l)cyanide (9.07 g, 101 .3 mmol) at room temperature. The mixture was stirred at 95 °C for 32 h. After cooling the mixture was and poured in a thoroughly stirred mixture of 5% sodium bicabonate solution (1 L) and ethyl acetate (500 mL) to give a black suspension. The mixture was filtered over Decalite®, flushed with warm ethyl acetate (2 x 150 mL). The organic layer from the biphasic filtrate was separated. The waterlayer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give 15.6 g of the crude title compound (yield: 78.9%)

[0324] (b) 6-Bromo-3-chloro-pyrazine-2-carbonitrile

[0325] To a solution of 3-amino-6-bromo-pyrazine-2-carbonitrile (15.6 g, 78.4 mmol) in acetonitrile (350 mL) was added copper(ll)chloride (12.65 g, 94.1 mmol) and the mixture was heated to 60 °C and stirred for 15 min. A solution of tert-butyl nitrite (1 1 .75 mL, 98 mmol) in acetonitrile (50 mL) was added dropwise at 60 °C to the reaction mixture. The mixture was stirred at 60 °C o / n. After cooling, the mixture was filtered over Decalite®, washed with acetonitrile. The filtrate was added dropwise to a 1 N HCI-solution (250 mL) and ethyl acetate (250 mL) and stirred for 10 min. The layers were separated and the waterlayer was extracted with ethyl acetate (2x 100 mL). The combined organic layers were washed with 1 N-HCl-solution, water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography (plug filtration) using SiO2and heptane / ethyl acetate = 95 / 5 to 9 / 1 to 8 / 2 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 10.4 g of the title compound (yield 60.7%).

[0326] (c) (6-Bromo-3-chloro-pyrazin-2-yl)methanamine hydrochloride

[0327] To a solution of 6-bromo-3-chloro-pyrazine-2-carbonitrile (10.4 g, 47.62 mmol) in acetic acid (160 mL) was added under nitrogen of Raney-Nickel (50% slurry in water, 1 .96 mL, ~16.7 mmol). The resulting mixture was shaken in a Parr-vessel under 4 bar hydrogen at room temperature o / n. Raney-Nickel was removed by filtration over Decalite® and the filtrate was concentrated under reduced pressure and co-evaporated with toluene. The remaining green solid (12.6 g) was suspended in ethyl acetate at 50 °C and filtered. The filtrate was cooled on an ice-bath, 2N HCIgasin diethyl ether (48 mL) was added dropwise and the resulting suspension was stirred at room temperature o / n. The solids formed were collected by filtration, washed with diethyl ether and dried under reduced pressure at 40 °C to give 6.21 g (50%) of (6-bromo-3- chloro-pyrazin-2-yl)methanamine hydrochloride (Intermediate 6).

[0328] Intermediate 7

[0329] Ethyl (E)-8-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)oct-7-enoate (Intermediate 7)

[0330] (a) Ethyl oct-7-ynoate

[0331] To a solution of oct-7-ynoic acid (7.92g, 56.53 mmol) in ethanol (70 ml) was added conc.- H2SO4(0.2 mL). The reaction mixture was stirred for 5 h. at 70 °C. After cooling of the mixture, MTBE (150 mL) was added and the organic phase was washed with sat. aq. NaHSO3-solution, water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give 8.84 g of ethyl oct-7-ynoate (yield: 93%).

[0332] (b) Ethyl ( E)-8-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)oct-7-enoate (Intermediate 7)

[0333] To a cold (5 °C) oven-dried round bottom flask equipped with a magnetic stirring bar were added ethyl oct-7-ynoate (9. 1 1g, 54.16 mmol), Schwartz’s reagent (1 .39 g, 5.41 mmol), Et3N (753 μL, 5.41 mmol) and pinacolborane (8.24 mL, 56.86 mmol), under an inert nitrogen atmosphere. The bottom flask was then sealed and the mixture was stirred at 60 °C for 5 hours. The reaction was allowed to cool to room temperature, diluted with dichloromethane, passed through a pad of silica gel and concentrated under reduced pressure at room temperature. The crude mixture was purified by column chromatography using SiO2and hexane / ethyl acetate = 99 / 1 to 9 / 1 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 6.42 g of the title compound (yield: 40%).

[0334] Intermediate 8 N-(4-Cvano-2-pyridyl)-4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzamide (Intermediate 8)

[0335] (a) 4-(4,4,5,5-Tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzoyl chloride

[0336] To a cold (0 °C) solution of 4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzoic acid (24.8 g, 100 mmol) in dichloromethane (300 mL) was added a catalytic amount of DMF. A solution of oxalyl chloride (12.9 mL, 150 mmol) was added dropwise. After stirring for 60 min at 0 °C, the reaction mixture was allowed to warm to room temperature and the mixture was stirred o / n. The reaction mixture was concentrated to give 26.33 g of crude 4-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)benzoyl chloride (yield: 99%).

[0337] (b) N-(4-Cvano-2-pyridyl)-4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzamide (Intermediate 8)

[0338] To a cold (0 °C) solution of 4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzoyl chloride (26.0 g, 100 mmol) in acetonitrile (300 mL) was subsequently added 2-aminopyridine- 4-carbonitrile (14.29 g, 120 mmol) and 4-DMAP (14.66 g, 120 mmol) The mixture was stirred under nitrogen atmosphere at 0°C and allowed to warm to room temperature overnight. The reaction mixture was concentrated in vacuo. The crude oily solids were then dissolved in dichloromethane (300 mL) and washed with 5% citric acid (3x, 300 mL), 5% NaHSO3(2x300 mL) and brine (200 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue triturated in acetonitrile / water = 1 / 1 v / v% (300 mL). The solids were filtrated and dried under reduced pressure to give 20.5 g of 4-(4,4,5,5-tetramethyl- 1 ,3,2-dioxaborolan-2-yl)-N-[4-(trifluoromethyl)-2-pyridyl]benzamide (Intermediate 8) (yield: 69%) as off-white solids.

[0339] Example A: Compound A Example A

[0340] (a) tert-Butyl N- [(1 R,3R)-3-[(6-bromo-3-chloro-pyrazin-2-yl)methylcarbamoyl]cyclohexyl]- carbamate

[0341] To a cold (4 °C) suspension of (1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexane- carboxylic acid (Intermediate 5, 10.36 g, 42.62 mmol) and (6-bromo-3-chloro-pyrazin-2- yl)methanamine hydrochloride (intermediate 6, 10.51 g, 40.59 mmol) in dichloromethane (406 mL) was added subsequently, DIPEA (21 .21 mL, 121 .77 mmol) and HATU (16.2 g, 42.62 mmol). The resulting mixture was stirred at room temperature o / n. The reaction mixture was filtered over a Buechner filter. The filtrate was washed with 5% aq. NaHSO3-solution (100 mL), 5% aq. citric acid solution (3x100 mL), brine (100 mL), dried over sodium sulfate filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2and heptane / ethyl acetate = 8 / 2 to 3 / 7 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 17.4 g of the title compound (Yield: 94%).

[0342] (b) Ethyl (E)-8-[6-[[[(1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexanecarbonyl]amino]methyl]- 5-chloro-pyrazin-2-yl]oct-7-enoate

[0343] A mixture of tert-butyl N-[(1 R,3R)-3-[(6-bromo-3-chloro-pyrazin-2-yl)methylcarbamoyl]- cyclohexyljcarbamate (17.4 g, 38.86 mmol), ethyl (E)-8-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan- 2-yl)oct-7-enoate (Intermediate 7, 1 1 .51 g, 38.86 mmol) and potassium carbonate (16.1 1 g, 1 16.6 mmol) in dioxane / water = 4 / 1 v / v% (200 mL) was degassed with nitrogen for 5 min at 30 °C. Pd(dppf)Cl2.CH2Cl2(1 .58 g, 1 .94 mmol) was added and the mixture was again degassed with nitrogen for 5 min at 30 °C. The reaction mixture was stirred at 80 °C for 4 h. After cooling ethyl acetate was added and the mixture was stirred for 15 min. The mixture was filtered over Decalite™ and the filtrate was washed with water and brine. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2and heptane / ethyl acetate = 7 / 3 to 3 / 7 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 20.9 g of the title compound (Yield: quantitative).

[0344] (c) Ethyl (E)-8-[3-[(1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexyl]-8-chloro-imidazo[1 ,5- alpyrazin-5-yl]oct-7-enoate

[0345] To a cold (-10 °C) solution of ethyl (E)-8-[6-[[[(1 R,3R)-3-(tert-butoxycarbonylamino)- cyclohexanecarbonyl]amino]methyl]-5-chloro-pyrazin-2-yl]oct-7-enoate (20.9 g, 38.9 mmol) in acetonitrile / DMF = 9 / 1 v / v% (1 17 mL) was added subsequently pyridine (12.58 mL, 155.7 mmol) and phosphorus oxychloride (7.25 mL, 77.84 mmol) and the mixture was stirred for 30 min. at 5 °C. The mixture was added carefully to 25% ammonia (200 mL) and crushed ice (400 mL) keeping the temperature below 0 °C. Ethyl acetate (200 mL) was added and the resulting mixture was stirred for 30 min. The water layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate and concentrated in vacuo to give 19.4 g of the title compound (yield: 96%). (d) Ethyl (E)-8-[1 -bromo-3-[(1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexyl]-8-chloro-imidazo- [1 ,5-alpyrazin-5-yl]oct-7-enoate N-Bromosuccinimide (7.31 g, 41 .1 mmol) was added to a cold (0 °C) solution of ethyl (E)-8-[3-[(1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexyl]-8-chloro-imidazo[1 ,5-a]pyrazin-5- yl]oct-7-enoate (19.4 g, 37.37 mmol) in acetonitrile (149 mL). The reaction mixture was stirred for 3 h. allowing the temperature to come to room temperature. The mixture was quenched with 10% aq. Na2S2O4-solution / 5% aq. NaHSO3-solution / brine and ethyl acetate. The phases were separated and the water layer was extracted with ethyl acetate. The combined organic phases were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2and heptane / ethyl acetate = 9 / 1 to 6 / 4 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 17.07 g of the title compound (Yield: 74%).

[0346] (e) Ethyl (E)-8-[1 -bromo-3-[(1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexyll-8-[(2,4- dimethoxyphenyl)methylamino1imidazo[1 ,5-alpyrazin-5-yl]oct-7-enoate

[0347] To a suspension of ethyl (E)-8-[1 -bromo-3-[(1 R,3R)-3-(tert-butoxycarbonylamino)- cyclohexyl]-8-chloro-imidazo-[1 ,5-a]pyrazin-5-yl]oct-7-enoate (17.07 g, 28.55 mmol) in 1 -butanol (1 14 mL) was added 2,4-dimethoxybenzylamine (12.86 mL, 85.65 mmol) and the mixture was stirred at 1 10 °C for 2 h. The mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate / water 5 / 2 v / v% (700 mL). The organic layers was separated and washed with 5% NaHSO3-solution (100 mL) and brine (2x50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2and heptane / ethyl acetate = 85 / 15 to 6 / 4 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 18.9 g of the title compound (Yield: 91 %).

[0348] (f) Ethyl (E)-8-[3-[(1 R,3R)-3-aminocyclohexyl1-1 -bromo-8-[(2,4-dimethoxyphenyl)methylamino1- imidazo[1 ,5-alpyrazin-5-yl]oct-7-enoate

[0349] Ethyl (E)-8-[1 -bromo-3-[(1 R,3R)-3-(tert-butoxycarbonylamino)cyclohexyl]-8-[(2,4-dime- thoxyphenyl)methylamino]imidazo[1 ,5-a]pyrazin-5-yl]oct-7-enoate (18.85 g, 25.86 mmol) was dissolved in dichloromethane (129 mL). Trifluoroacetic acid (19.2 mL) was added and the mixture was stirred at rroooomm temperature for 2 h. The mixture was poured into dichloromethane / water = 1 / 4 v / v% (750 mL), the water layer was separated and extracted with dichloromethane (100 mL). The combined organic layers were washed with sat. aq. NaHSO3- solution (100 mL), brine (50 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give 16.68 g of the title compound (Yield: 102%).

[0350] (g) Lithium (E)-8-[3-[(1 R,3R)-3-aminocyclohexyll-1 -bromo-8-[(2,4-dimethoxvphenyl)methyl- aminolimidazofl ,5-alpyrazin-5-yl]oct-7-enoate

[0351] To a solution ethyl (E)-8-[3-[(1 R,3R)-3-aminocyclohexyl]-1 -bromo-8-[(2,4-dimethoxy- phenyl)methylamino]-imidazo[1 ,5-a]pyrazin-5-yl]oct-7-enoate (16.68 g, 25.86 mmol) in THF / water=4 / 1 v / v% (129 mL) was lithium hydroxide (648 mg, 27.15 mmol) and the mixture was stirred at room temperature for 72 h. Water (300 mL) was added to the mixture and after evaporation of THF, the mixture was lyophilized to give 15.9 g of the title compound (yield: quantitative crude).

[0352] (h) Macrocyclic Intermediate 9

[0353] Lithium (E)-8-[3-[(1 R,3R)-3-aminocyclohexyl]-1 -bromo-8-[(2,4-dimethoxyphenyl)- methylamino]imidazo[1 ,5-a]pyrazin-5-yl]oct-7-enoate (9.14 g, 15.07 mmol) was suspended dissolved in DMF (520 mL) and N-ethylmorpholine (3.83 mL, 30.14 mmol) was added. This solution was added slowly to a heavily stirred suspension of HATU (17.18 g, 45.21 mmol) and N-ethylmorpholine (5.74 mL, 45.21 mmol) in ethyl acetate (1 .0 L) over a 1 1 h period to obtain a clear solution. The mixture was poured into stirred cold water (2.0 L) and stirring was continued for 15 min. The water layer was separated and extracted with ethyl acetate / heptane = 95 / 5 v / v% (3x300 mL). The combined organic layer was washed with washed with 2.5% aq. citric acid solution (300 mL), sat. aq. NaHSO3-solution (300 mL), water (300 mL) and brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using SiO2and heptane / ethyl acetate / methanol = 50 / 50 / 0 to 0 / 92.5 / 7.5 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 7.39 g of the DMB-protected Macrocyclic Intermediate 9 (Yield: 84%).

[0354] (I) Example A: Compound A

[0355] DMB-protected Macrocyclic Intermediate 9 (9.83 g, 16.87 mmol) was dissolved in TFA (67 mL) and the mixture was stirred at 60 °C for 4 h. The reaction mixture was added slowly to a mixture of dichloromethane / water = 1 / 1 v / v% (1 .0 L). The bi-phasic mixture was stirred for 10 min. The layers were separated and the water layer was extracted with dichloromethane (200 mL). To the combined organic layer was added slowly under stirring 12.5% NFUOH (300 mL). The layers were separated and the organic layer was washed with 12.5% NFUOH (150 mL), water (250 mL) and brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was triturated with hot (90 °C) acetonitrile (100 mL), filtered and dried to give 5.9 g (yield: 80%) of the deprotected Macrocyclic Intermediate 9 which was used directly in the next step.

[0356] Deprotected Macrocyclic Intermediate 9 (2.25 g, 5.2 mmol) was dissolved in dioxane / water = 9 / 1 v / v% (100 mL) and potassium carbonate (2.15 g, 15.6 mmol) was added. The solution was purged with nitrogen for 5 min and N-(4-cyano-2-pyridyl)-4-(4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzamide (Intermediate 8) (2.18 g, 6.24 mmol) and Pd(dppf)Cl2.CH2Cl2(424 mg, 0.52 mmol) were added. The reaction mixture was stirred for 4 h at 90 °C. The reaction mixture was diluted with dichloromethane / methanol = 9 / 1 v / v% (400 mL) after cooling to room temperature and filtered over Decalite™. The filtrate was washed with water, and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2and dichloromethane / methanol = 98 / 2 to 94 / 6 v / v%. All fractions containing the title compound were collected and concentrated in vacuo to give 1 .6 g of the title compound (Yield: 53%). Data: LCMS (B) Rt: 6.99 min; m / z 575.4 [M+H]+.

[0357] Remark: Compound A as described herein is the same compound as compound having subformula 184 of co-pending PCT application PCT / EP2022 / 085765. Said compound having subformula 184 is also incorporated by reference.

[0358] Examples

[0359] Cell culture

[0360] TMD8 diffuse large B-cell lymphoma cells were purchased from Tokyo Medical and Dental University. TMD8 cells expressing mutant BTK were created at Synthego Corporation. TMD8 cell lines expressing BTK C481S, BTK T474I, BTK T474I / C481S, BTK V416L and BTK L528W were generated via CRISPR / Cas9. BTK T474I / C481S expressing cells were generated by re- editing BTK C481S cells. TMD8 mutant cell pools were enriched to obtain a 100% mutant genotype. The mutation status of all cell lines was confirmed via sequencing. Cells were cultured in MEM-alpha cell culture medium (cat. no. 22571038, Life Technologies), supplemented with 10% (v / v) heat-inactivated fetal bovine calf serum (Avantor, cat. no. 97068085) and 1 % penicillin / streptomycin.

[0361] Proliferation assays

[0362] Inhibition of proliferation in response to compound was measured using the ATPIite 1 Step™ assay (cat. no. 6016736, Perkin Elmer). The assay utilizes ATP as a marker for metabolically active cells. A decline in ATP concentration indicates that compounds exert anti-proliferative effects on the cells under investigation, compared to vehicle treated cells. Measurement of ATP is based on the production of light caused by the reaction of ATP with added luciferase and D- luciferin. Luminescence was recorded using an Envision multilabel reader (Perkin Elmer). The ATP signal of the cells at the start of incubation (t=0) was recorded separately to distinguish between cell growth and cell death. Maximum growth was determined via vehicle treated cells, which indicates 100% viability (vehicle control). Percentage viability was used as the main y-axis signal. IC50s were fitted by non-linear regression using IDBS XLfit™ 5 using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50. In addition to the IC50, efficacy, Gl50and LD50were also determined.

[0363] Proliferation assays on BTK single mutant cell lines

[0364] WT-BTK, BTK C481S, BTK T474I, BTK T474I / C481S, BTK V416L and BTK L528W TMD8 cell lines were seeded at 800 cells per well in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for at least 4 hours at 37 °C, 95 % humidity, and 5 % CO2. Compound A, pirtobrutinib, ibrutinib, acalabrutinib and nemtabrutinb were diluted in 3.16-fold dilution steps in 100% DMSO and further diluted in 20 mM Hepes, generating a dose response range from 316 nM to 0.0316 nM in the assay. DMSO was used as a vehicle control.

[0365] Luminescence was recorded at 96 hours.

[0366] Combination experiments of Compound A with ibrutinib or Compound A with acalabrutinib in

[0367] BTK C481S TMD8 cells

[0368] BTK C481S TMD8 cells were seeded at 800 cells per well in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for at least 4 hours at 37 °C, 95 % humidity, and 5 % CO2. Compound A was diluted in 3.16-fold dilution steps in 100% DMSO and further diluted in 20 mM Hepes, generating a dose response range from 316 nM to 0.0316 nM in the assay. Ibrutinib or acalabrutinib was added in a fixed, inactive concentration to each concentration of Compound A, ranging from 316 nM to 0.316 nM. DMSO was used as a vehicle control. Luminescence was recorded at 96 hours. Anti-proliferative responses were assessed by 1 ) determining IC50shifts of the combinations at 96 hours compared to the single compounds, in which an leftward IC50shift of factor 2 is indicative of synergy; 2) calculating excess over Bliss scores (Lui et al. 2018).

[0369] Combination experiments with BTK C481S and BTK-mutant TMD8 cells

[0370] The BTK C481S TMD8 cell line was combined with either BTK T474I, BTK V416L or BTK L528W TMD8 cell lines and seeded at 800 cells per well for each cell line (1600 cells / well total) in white 384-well culture plates (cat. no. 781080, Greiner Bio-One). Cells were allowed to rest for at least 4 hours at 37 °C, 95 % humidity and 5 % CO2. Ibrutinib or acalabrutinib were diluted in 3.16-fold dilution steps in 100% DMSO and further diluted in 20 mM Hepes, generating a 9-point dose response range from 316 nM to 0.0316 nM in the assay. Compound A was added in a fixed concentration to each concentration of ibrutinib or acalabrutinib, ranging from 316 nM to 0.316 nM. DMSO was used as a vehicle control. Luminescence was recorded at 96 hours. Anti- proliferative responses were assessed 1 ) calculating efficacy of the single compounds and the combination compounds, compared to vehicle control at 96h, and 2) calculating the gain in efficacy of the combination compared to the single compounds.

[0371] Surface plasma resonance

[0372] Binding kinetics measurements on wt-BTK, BTK C481S, BTK T474I (Surface Plasmon Resonance) Streptavidin-coated chips (Cat. No. BR100531 ), disposables and maintenance kits for Biacore were purchased from Cytiva (Eindhoven, The Netherlands). Biotinylated BTK WT enzym (Carna Biosciences, cat. no. 08-480-20N), BTK C481S (Carna Biosciences, cat. no. 08- 417-20N), BTK T474I (Carna Biosciences, cat. no. 08-419-20N) were immobilized on a streptavidin-coated chip to a level of about 8000 resonance units (RU) using Biacore buffer (50 mM Tris pH 7.5, 0.05 % (v / v) Tween-20, 150 mM NaCI and 5 mM MgCI2) + 1 mM TCEP. 10 Remaining streptavidin was blocked with biocytin. Immobilization was performed at 4°C. Subsequent assay steps were conducted at 22°C. After changing buffer to Biacore buffer with 1 % (v / v) dimethylsulfoxide (DMSO), a pre-run was performed for a period of at least 30 min at a flow rate of 30 μl / min to obtain a stable surface. The kinetic constants of the compounds were determined with single cycle kinetics with five consecutive injections with an increasing 15 compound concentration with ranges of 3.16 - 316 nM. Experiments were performed with an association time of 100 s per concentration and a dissociation time of 1200 s, except for compounds with a long target residence time, such as irreversible inhibitors, where dissociation time was increased. To circumvent problems of mass transport limitation, a flow rate of 30 μl / min was used. A blank run with the same conditions was performed before the compound was 20 injected. The SPR sensorgrams were analyzed with Biacore Evaluation Software by using a method of double referencing. First the reference channel was subtracted from the channel containing immobilized protein. Subsequently, the reference curve obtained with buffer injections was subtracted. The resulting curve was fitted with a 1 :1 binding model. Compounds that bound according to an induced fit model were fitted with a two-state reaction model. The kinetic 25 constants (ka, kd, KD) of duplicates were geometrically averaged. Target residence time (T) for the 1 :1 binding model was calculated from the dissociation constant kdwith the formula τ = 1 / kd. Target residence for an induced fit model was calculated as described (Tummino and Copeland, 2008).

[0373] Results

[0374] Proliferation assay of single compounds (monotherapy) in wt-BTK TMD8 cells (Figure 2)

[0375] Viability of Compound A and other compounds in wt-BTK TMD8 cells. All compounds inhibit proliferation with <10 nM IC50s and >90% efficacy, nemtabrutinib excluded. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

[0376] Table 1; Proliferation data for wt-BTK TMD8 cells

[0377]

[0378] Proliferation assay of single compounds (monotherapy) in BTK C481S TMD8 cells. (Figure 3)

[0379] Viability of Compound A and other compounds in BTK C481S TMD8 cells. Compound A, pirtobrutinib and nemtabrutinib inhibit proliferation. Ibrutinib and acalabrutinib do not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

[0380] Table 2; Proliferation data for BTK C481S TMD8 cells

[0381] Proliferation assay of single compounds (monotherapy) in BTK T474I TMD8 cells. (Figure 4)

[0382] Viability of Compound A and other compounds in BTK T474I TMD8 cells. Compound A, ibrutinib and acalabrutinib inhibit proliferation. Pirtobrutinib and nemtabrutinib do not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

[0383] Table 3: Proliferation data for BTK T474I TMD8 cells

[0384]

[0385] Proliferation assay of single compounds (monotherapy) in BTK T474I / C481S TMD8 cells.

[0386] (Figure 5)

[0387] Viability of Compound A and other compounds in BTK T474I / C481S TMD8 cells. Only Compound A inhibits proliferation. All other compounds are unable to inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

[0388] Table 4; Proliferation data for BTK T474I / C481S TMD8 cells

[0389] Proliferation assay of single compounds (monotherapy) in BTK V416L TMD8 cells. (Figure 6)

[0390] Viability of Compound A and other compounds in BTK V416L TMD8 cells. Compound A, ibrutinib and acalabrutinib inhibit proliferation. Pirtobrutinib and nemtabrutinib do not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

[0391] Table 5: Proliferation data for BTK V416L TMD8 cells

[0392] Proliferation assay of single compounds (monotherapy) in BTK L528W TMD8 cells. (Figure 7)

[0393] Viability of Compound A and other compounds in BTK L528W TMD8 cells. Compound A and acalabrutinib inhibit the proliferation. Ibrutinib and pirtobrutinib show low efficacy and nemtabrutinib does not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

[0394] Table 6: Proliferation data for BTK L528W TMD8 cells

[0395] Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells (Figure 8)

[0396] Viability of ibrutinib, acalabrutinib and pirtobrutinib in a 1 :1 mixture of BTK C481S TMD8 cells + BTK V416L TMD8 cells after 96h incubation. Graphs represent efficacy of compounds used in the standard of care. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the highest tested concentration from vehicle treated cells. The dotted line indicates the cell population at the start of the experiment (t=0). Table 7; Proliferation data for compounds in a mixture of BTK C481S TMD8 cells + BTK V416L TMD8 cells.

[0397] Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells (Figure 9)

[0398] Viability of ibrutinib, acalabrutinib and pirtobrutinib in a 1 :1 mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells after 96h incubation. Graphs represent efficacy of compounds used in the standard of care. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the highest tested concentration from vehicle treated cells. The dotted line indicates the cell population at the start of the experiment (t=0).

[0399] Table 8; Proliferation data for compounds in a mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells.

[0400] Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells (Figure 10)

[0401] Viability of ibrutinib, acalabrutinib and pirtobrutinib in a 1 :1 mixture of BTK C481S TMD8 cells + BTK L528W TMD8 cells after 96h incubation. Graphs represent efficacy of compounds used in the standard of care. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the highest tested concentration from vehicle treated cells. The dotted line indicates the cell population at the start of the experiment (t=0).

[0402] Table 9; Proliferation data for compounds in a mixture of BTK C481S TMD8 cells + BTK L528W TMD8 cells.

[0403] COMBINATION THERAPY - IBRUTINIB - Compound A

[0404] Proliferation assay of ibrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells (Figure 11 )

[0405] Viability of ibrutinib, 31.6 nM Compound A and the combination of ibrutinib and 31.6 nM Compound A in a 1 :1 mixture of BTK C481S TMD8 cells + BTK V416L TMD8 cells after 96h incubation. In the combination, each concentration of ibrutinib contained the indicated concentration of Compound A. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM ibrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

[0406] Table 10; Proliferation data for a combination of ibrutinib and Compound A in a mixture of BTK C481S TMD8 cells + BTK V416L TMD8 cells.

[0407] Proliferation assay of ibrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells (Figure 12)

[0408] Viability of ibrutinib, 100 nM Compound A, and the combination of ibrutinib and 100 nM Compound A in a 1 :1 mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells after 96h incubation. In the combination, each concentration of ibrutinib contained the indicated concentration of Compound A. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM ibrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

[0409] Table 11; Proliferation data for a combination of ibrutinib and Compound A in a mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells.

[0410] Proliferation assay of ibrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells (Figure 13)

[0411] Viability of ibrutinib, 100 nM Compound A, and the combination of ibrutinib and 100 nM Compound A in a 1 :1 mixture of BTK C481S TMD8 cells + BTK L528W TMD8 cells after 96h incubation. In the combination, each concentration of ibrutinib contained the indicated concentration of Compound A. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM ibrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

[0412] Table 12; Proliferation data for a combination of ibrutinib and Compound A in a mixture of BTK C481S TMD8 cells + BTK L528W TMD8 cells.

[0413] COMBINATION THERAPY - Acalabrutinib - Compound A

[0414] Proliferation assay of acalabrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells (Figure 14)

[0415] Viability of acalabrutinib, 100 nM Compound A, and the combination of acalabrutinib and 100 nM Compound A in a 1 :1 mixture BTK C481S TMD8 cells + BTK V416L TMD8 cells after 96h incubation. In the combination, each concentration of acalabrutinib contained the indicated concentration of Compound A. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM acalabrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

[0416] Table 13; Proliferation data for a combination of acalabrutinib and Compound A in a mixture of BTK C481S TMD8 cells + BTK V416L TMD8 cells.

[0417] Proliferation assay of acalabrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells (Figure 15)

[0418] Viability of acalabrutinib, 31 .6 nM Compound A, and the combination of acalabrutinib and 31 .6 nM Compound A in a 1 :1 mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells after 96h incubation. In the combination, each concentration of acalabrutinib contained the indicated concentration of Compound A. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells.

[0419] Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM acalabrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

[0420] Table 14; Proliferation data for a combination of acalabrutinib and Compound A in a mixture of

[0421] BTK C481S TMD8 cells + BTK T474I TMD8 cells.

[0422] Proliferation assay of acalabrutinib and Compound A (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells (Figure 16)

[0423] Viability of acalabrutinib, 31 .6 nM Compound A, and the combination of acalabrutinib and 31 .6 nM Compound A in a 1 :1 mixture of BTK C481S TMD8 cells + BTK L528W TMD8 cells after 96h incubation. In the combination, each concentration of acalabrutinib contained the indicated concentration of Compound A. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells.

[0424] Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM acalabrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

[0425] Table 15; Proliferation data for a combination of acalabrutinib and Compound A in a mixture of

[0426] BTK C481S TMD8 cells + BTK L528W TMD8 cells. COMBINATION THERAPY -Compound A - IBRUTINIB

[0427] Proliferation assay of ibrutinib and Compound A (combination therapy) in BTK C481S

[0428] TMD8 cells

[0429] Compound A was tested in combination with fixed concentrations of ibrutinib in BTK C481S TMD8 cells, see Figure 17A - 17G.

[0430] Viability and potency of Compound A, ibrutinib, and combinations of Compound A and ibrutinib. IC50shifts were determined by comparing the individual IC50s of Compound A and ibrutinib with the IC50of the combination. In the combination each concentration of compound A contained the indicated concentration of ibrutinib. A leftward IC50shift of factor >2 is indicative of synergy (pIC50(M) of >0.30).

[0431] Table 16; Proliferation data for a combination of ibrutinib and Compound A in BTK C481S TMD8 cells (Curve shift analysis).

[0432] Excess over Bliss score matrix of Compound A and ibrutinib in BTK C481S TMD8 cells, see Figure 18.

[0433] Excess over Bliss scores of the combination of Compound A and ibrutinib in BTK C481S TMD8 cells after 96h incubation. Concentrations in nM of compound A are depicted horizontally, concentrations of ibrutinib vertically. Number in italic represent the viability of the single compounds relative to untreated at the indicated concentrations.

[0434] COMBINATION THERAPY -Compound A - acalabrutinib

[0435] Proliferation assay of acalabrutinib and Compound A (combination therapy) in BTK C481S TMD8 cells Compound A was tested in combination with fixed concentrations of acalabrutinib in BTK C481S TMD8 cells, see Figures 19A - 19G.

[0436] Viability and potency of Compound A, acalabrutinib, and combinations of Compound A and acalabrutinib. IC50shifts were determined by comparing the individual IC50s of Compound A and acalabrutinib with the IC50of the combination. In the combination each concentration of compound A contained the indicated concentration of acalabrutinib. A leftward IC50shift of factor >2 is indicative of synergy (pIC50(M) of >0.30).

[0437] Table 17; Proliferation data for a combination of acalabrutinib and Compound A in BTK C481S TMD8 cells (Curve shift analysis).

[0438] Excess over Bliss score matrix of Compound A and acalabrutinib in BTK C481S TMD8 cells, see Figure 20.

[0439] Excess over Bliss scores of the combination of Compound A and acalabrutinib in BTK C481S TMD8 cells after 96h incubation. Concentrations in nM of compound A are depicted horizontally, concentrations of acalabrutinib vertically. Number in italic represent the viability of the single compounds relative to untreated at the indicated concentrations.

[0440] The results show that the combination of Compound A and ibrutinib or the combination of Compound A and acalabrutinib leads to increased potency and efficacy in BTK C481S TMD8 cells. The high excess over Bliss scores and leftwards IC50shifts at low concentrations of both Compound A and ibrutinib or Compound A and acalabrutinib indicate synergy. This provides excellent opportunities for combination therapies in subjects that have developed or are at risk of developing resistance.

[0441] Surface plasma resonance Target residence times for compound A and reference reversible inhibitors are given in Table 18 below.

[0442] Table 18: KD and Target residence times (T (h)) for BTK inhibitors on wt-BTK, BTK C481S, BTK T474I measured with SPR.

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Claims

Claims1 . Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, the method comprising: administering to the subject a therapeutic combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor, wherein the reversible BTK inhibitor is a compound of Formula (l-a) to (l-h) or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound is selected from the group consisting of:(Formula l-a) (Formula l-b) (Formula l-c) (Formula l-d)(Formula l-e) (Formula l-f) (Formula l-g) (Formula l-h)Wherein R1is , wherein :W is an aryl group having 6-10 carbon or a heteroaryl group having 1 -5 carbon; wherein any said aryl group and heteroaryl group is optionally and independently substituted with one or more substituents selected from halogen, (1 -2C)alkyl, (1 -2C)alkoxy; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three fluoro;V is any one of O, -C(O)-NH-, -NH-C(O)-, -CH(R1v)-NH-C(O)-, -CH(R1v)- ;R1vis hydrogen or (1 -2C)alkyl;U is an aryl group having 6-10 carbon or an heteroaryl group having 1 -5 carbon; wherein any of said aryl group and heteroaryl group is optionally and independently substituted with one or more substituents selected from halogen, cyano, (1 -4C)alkyl, (1 -5C)alkoxy, (3-6C)cycloalkyl or (3-6C)heterocycloalkyl; wherein any of said alkyl, alkoxy, cycloalkyl and heterocycloalkyl group is optionally and independently substituted with one, two or three halogen; wherein R2is of Formula (ll-a) to (ll-f) selected from the group consisting of:10(Formula ll-a) (Formula ll-b) (Formula ll-c)(Formula ll-d) (Formula ll-e) (Formula ll-f) wherein Q is a monocyclic ring selected from a (3-7C)cycloalkyl and a (3- 6C)heterocycloalkyl, wherein Xi , X2 and X3 are independently selected from CH2, - CH2CH2-, O, N and a direct bond; wherein any of the cycloalkyl, heterocycloalkyl and alkyl group is optionally and independently substituted with one or more substituents selected from halogen, hydroxy, (1 -3C)alkyl, (1 -3C)alkoxy, (1 -4C)alkylcarbonyl or (3- 4C)cycloalkyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three halogen; wherein R3and R4together represent a linker having Formula (111-1 to III-40) selected from the group consisting of:30n = 0 - 6 n = 1 - 5 n = 0 - 4 n = 0 - 4 n = 1 - 5(111-1 ) (111-2) (111-3) (111-4) (111-5)510whereby themarks the position of R3in any one of Formula l-a to l-h, and whereby themarks the position of R4in any one of Formula I l-a to ll-f;wherein any of said linkers is optionally and independently substituted with one or more substituents selected from deuterium, halogen, oxo, hydroxy, CD3, (1 -4C)alkyl, (1 - 5C)alkoxy, (3-6C)cycloalkyl, (3-6C)cycloalkoxy and (1 -6C)alkylcarbonyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three halogen.

2. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject diagnosed with a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has previously been treated with an irreversible BTK inhibitor, and wherein the method comprises administering to said subject a therapeutic combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor, wherein the reversible BTK inhibitor is a compound of any one of Formula (l-a) to (l-h) as defined in claim 1 .

3. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, the method comprising the steps:(a) monitoring the subject, preferably at predetermined intervals of time, over the course of the therapy for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in resistance or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor of the (mono) therapy, preferably the subject has a mutation in an endogenous gene encoding BTK that results in expression of a BTK protein with a modification at an amino acid position corresponding to amino acid position 481 of the amino acid sequence according to SEQ ID NO: 1; and(b) if the subject has shown said mutation, administering to the subject a therapeutically combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor, in particular if the subject has the BTK modification corresponding to amino acid position 481 identified in step (a), wherein the reversible BTK inhibitor is a compound of any one of Formula (l-a) to (l-h) as defined in claim 1 .

4. Combination for use in the method of any one of claims 1 -3, wherein the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, spebrutinib, branebrutinib, evobrutinib, remibrutinib, tolebrutinib,orelabrutinib, elsubrutinib, edralbrutinib, ACP-5862 and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

5. Combination for use in the method of claim 1 or 4, wherein the subject has been administered a BTK inhibitor for treatment of the hyperproliferative disease, preferably the B-cell hematological malignancy, preferably before recurrence or relapse of the hyperproliferative disease.

6. Combination for use in the method of any one of the preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: 1, more preferably wherein the protein modification is C481S.

7. Combination for use in the method of any one of the preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: 1, wherein the modification is a substitution of cysteine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 481 of the BTK protein.

8. Combination for use in a method of claim 7, wherein the modification is a substitution of cysteine to serine at amino acid position 481 of the BTK protein.

9. Combination for use in a method of any one of preceding claims, wherein the reversible BTK inhibitor is a compound according to Formula (l-a) or (l-b) or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound comprises a bicyclic scaffold selected from:(Formula l-a) (Formula l-b)Wherein R1is any one of:5wherein R2wis selected from hydrogen, halogen, (1 -2C)alkyl, (1-2C)alkoxy; wherein any said alkyl or alkoxy group is optionally and independently substituted with one, two or three fluoro; wherein R3uis selected from hydrogen, halogen, cyano, (1 -4C)alkyl, (1 -5C)alkoxy, (3- 6C)cycloalkyl or (3-6C)heterocycloalkyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three fluoro; wherein R2is selected from the group consisting of:20(Formula Il-a3) (Formula Il-a4) (Formula Il-b3) (Formula Il-c3)25(Formula ll-d) (Formula ll-f) (Formula Il-a5) (Formula Il-b4) wherein any of said cycloalkyl, heterocycloalkyl and alkyl group is optionally and independently substituted with hydroxy, methyl, acetyl or methoxy; wherein R3and R4together represent a linker having Formula selected from the group consisting of:whereby themarks the position of R3in any one of Formula l-a to l-b, and whereby themarks the position of R4in any one of Formula I l-a to I l-f; wherein any of said linkers is optionally and independently substituted with one or more substituents selected from deuterium, halogen, oxo, hydroxy, CD3, (1 -4C)alkyl, (1 - 5C)alkoxy, (3-6C)cycloalkyl, (3-6C)cycloalkoxy and (1-6C)alkylcarbonyl; wherein any of said alkyl and alkoxy group is optionally and independently substituted with one, two or three halogen.

10. Combination for use in a method of any one of preceding claims, wherein the reversible BTK inhibitor is a compound according to Formula A:(A), or pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.1 1 . Combination for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

12. Combination for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is ibrutinib, or pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

13. Combination for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is acalabrutinib, or pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

14. Combination for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is zanubrutinib, or pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

15. Combination for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is tirabrutinib, or pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

16. Combination for use in a method of any one of preceding claims, wherein the subject is identified as having one or more mutations in a gene that results in expression of a BTKprotein with a modification at one or more amino acid positions selected from the group consisting of: 416, 428, 437, 474, 481 and 528.

17. Combination for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 416 of the amino acid sequence according to SEQ ID NO: 1, more preferably wherein the mutant modification is V416L.

18. Combination for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 428 of the amino acid sequence according to SEQ ID NO: 1, more preferably wherein the mutant modification is A428D.

19. Combination for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 437 of the amino acid sequence according to SEQ ID NO: 1, more preferably wherein the mutant modification is M437R.

20. Combination for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 474 of the amino acid sequence according to SEQ ID NO: 1, more preferably wherein the mutant modification is T474I.21 . Combination for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein at amino acid position 528 of the amino acid sequence according to SEQ ID NO: 1, more preferably wherein the mutant modification is L528W.

22. Combination for use in a method of any one of preceding claims, wherein the subject is monitored at predetermined intervals of time over the course of the therapy for treatment of the hyperproliferative disease, wherein the predetermined interval of time is every week, every 2 weeks, every 3 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 1 1 months, every year following the first administration of the irreversible BTK inhibitor.

23. Combination for use in a method of any one of preceding claims, wherein the irreversibleBTK inhibitor of the combination is administered in an amount in the range of 70 - 750 mg / day and / or wherein the irreversible BTK inhibitor of the combination is administered using one or more unit doses having an amount in the range of 70 - 750 mg / unit dose.

24. Combination for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is administered daily at a dose selected from the group consisting of 70 mg, 100 mg, 140 mg, 160 mg, 200 mg, 280 mg, 320 mg, 420 mg, 480 mg, and 560 mg per day and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

25. Combination for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is administered twice daily using a unit dose of 80 mg, 100 mg or 160 mg and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

26. Combination for use in a method of any one of preceding claims, wherein the dose amount of the irreversible BTK inhibitor of the therapeutically combinatory amount of the combination is lower than the dose amount of the same irreversible BTK inhibitor administered during the prior therapy.

27. Combination for use in a method of any one of preceding claims, wherein the therapeutically combinatory amount of the dose of the irreversible BTK inhibitor and the dose of the reversible BTK inhibitor of the combination is lower than the dose of the irreversible BTK inhibitor, which was administered during the prior therapy.

28. Combination for use in a method of any one of preceding claims, wherein the hyperproliferative disease is a B-cell hematological malignancy.

29. Combination for use in a method of any one of preceding claims, wherein the B-cell hematological malignancy is any one of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCB DLBCL), primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma, Burkitt’s lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B- lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma / Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

30. Combination for use in a method of any one of preceding claims, wherein the B-cell hematological malignancy is any one of mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), Waldenstrom macroglobulinemia (WM), follicular lymphoma (FL) and marginal zone lymphoma (MZL).31 . Combination for use in a method of any one of preceding claims, wherein the B-cell malignancy is Mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL).

32. Combination for use in a method of any one of claim 28 - 31 , wherein the B-cell hematological malignancy of the subject is relapsed or refractory, preferably wherein CLL / SLL is relapsed or refractory.

33. Combination for use in a method of any one of claim 28 - 32, wherein the B-cell hematological malignancy of the subject is relapsed or refractory and the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 , of the amino acid sequence according to SEQ ID NO: 1, more preferably wherein the mutant modification is C481S.

34. Combination for use in a method of any one of the preceding claims, wherein the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in a combined dosage form.

35. Combination for use in a method of any one of the preceding claims, wherein the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in separate dosage forms.

36. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, wherein the reversible BTK inhibitor is a compound of any one of Formula (l-a) to (l-h) as defined in any one of claim 1 , claim 9 or claim 10.

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